Patent application title: MICROBIOLOGICAL PRODUCTION OF SHORT FATTY ACIDS AND USES THEREOF
Inventors:
Martin Grininger (Frankfurt, DE)
Jan Gajewski (Frankfurt, DE)
Eckhard Boles (Darmstadt, DE)
Eckhard Boles (Darmstadt, DE)
Renata Pavlovic (Zedge, SG)
IPC8 Class: AC12N910FI
USPC Class:
1 1
Class name:
Publication date: 2021-12-23
Patent application number: 20210395703
Abstract:
The present invention relates to proteins involved in fatty acid
synthesis, such as fatty acid synthases (FAS) variants, comprising one or
more polypeptide chains, wherein said polypeptide chain(s) comprise one
or more subunits comprising a malonyl/palmitoyl transferase domain (MPT
domain), acetyl transferase domain (AT domain), and ketoacyl synthase
domain (KS domain), and at least one amino acid substitution in the MPT
domain at a position corresponding to R130, in the AT domain at a
position corresponding to 1306, and/or in the KS domain, preferably in
the acyl binding channel and/or at KS domain binding site to ACP, to
modulate affinities of acyl intermediates, and optionally further amino
acid substitution(s). The present invention relates to the respective
polypeptide domains.
The present invention further relates to nucleic acid molecules encoding
the proteins (or the polypeptide domains) and to host cells containing
said nucleic acid molecules. The present invention further relates to a
method for the production of short fatty acids, CoA esters of short fatty
acids, ethyl esters of short fatty acids, esters of short fatty acids
with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to
C.sub.12), comprising the expression of said nucleic acid molecules,
preferably in said host cells. The present invention further relates to a
method for the production of biofuels, flavoring compounds and/or fine
chemicals, comprising the expression of said nucleic acid molecules,
preferably in said host cells. The present invention also relates to the
use of the proteins, nucleic acids molecule or host cells for the bulk
production of short fatty acids (C.sub.6 to C.sub.12), the specific
production of C.sub.6 fatty acids and/or C.sub.8 fatty acids, the bulk
production of CoA esters of short fatty acids (C.sub.6 to C.sub.12), the
specific production of C.sub.6-CoA esters and/or C.sub.8-CoA esters, the
bulk production of ethyl esters of short fatty acids (C.sub.6 to
C.sub.12), the specific production of C.sub.6 fatty acid ethyl esters
and/or C.sub.8 fatty acid ethyl esters, the bulk production of esters of
short fatty acids (C.sub.6 to C.sub.12) with other metabolites, the
specific production of C.sub.6 fatty acid esters with other metabolites
and/or C.sub.8 fatty acid esters with other metabolites, the bulk
production of enzyme bound short fatty acids (C.sub.6 to C.sub.12), the
specific production of enzyme bound C.sub.6 fatty acids and/or enzyme
bound C.sub.8 fatty acids, the production of biofuels, fine chemicals
and/or flavoring substances.Claims:
1. A protein involved in fatty acid synthesis, said protein comprising
one or more polypeptide chains, wherein said polypeptide chain(s)
comprise (i) one or more subunits comprising the amino acid sequences of
SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain); SEQ ID
NO: 2 (acetyl transferase domain, AT domain), and/or SEQ ID NO: 3
(ketoacyl synthase domain, KS domain); (ii) at least one amino acid
substitution in the MPT domain at a position corresponding to R130 of the
amino acid sequence of SEQ ID NO: 1; in the AT domain at a position
corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2; and/or
in the KS domain, in the acyl binding channel; wherein the amino acid
sequence comprising the at least one amino acid substitution has at least
80% sequence identity to the respective amino acid sequence of SEQ ID NO:
1 and/or 2, and/or at least 80% sequence identity to the respective amino
acid sequence of SEQ ID NO: 3, provided that when the protein comprises
an amino acid substitution G236S in the KS domain it comprises at least
one additional amino acid substitution.
2. The protein according to claim 1, comprising at least one further amino acid substitution in the KS domain selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3.
3. The protein according to claim 1, wherein the protein is type I FAS of Saccharomyces cerevisiae.
4. A polypeptide domain comprising (i) one or more subunits comprising the amino acid sequences of SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain); SEQ ID NO: 2 (acetyl transferase domain, AT domain), or SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); (ii) at least one amino acid substitution in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1; in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2; and/or in the KS domain in the acyl binding channel; wherein the amino acid sequence comprising the at least one amino acid substitution has at least 80% sequence identity to the respective amino acid sequence of SEQ ID NO: 1 and/or 2, and/or at least 80% sequence identity to the respective amino acid sequence of SEQ ID NO: 3, provided that when the polypeptide domain comprises an amino acid substitution G236S in the KS domain it comprises at least one additional amino acid substitution.
5. The polypeptide domain according to claim 4, comprising at least one further amino acid substitution in the KS domain selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3.
6. The polypeptide domain according to claim 1, comprising amino acid substitution(s) R130K in the MPT domain (SEQ ID NO. 1), and/or I306A in the AT domain (SEQ ID NO: 2).
7. The polypeptide domain according to claim 1, comprising amino acid substitution(s) in the acyl binding channel of the KS domain, wherein the amino acid substitutions comprise amino acid substitution(s) G236S, M237W and/or F265Y in the KS domain (SEQ ID NO: 3), and/or further comprising amino acid substitution(s) Q193A, Q193E, N258A, N258D and/or D259A in the KS domain (SEQ ID NO: 3).
8. The protein according to claim 1, selected from the group of a protein comprising the amino acid substitutions I306A and G236S; a protein comprising the amino acid substitutions I306A, R130K and F265Y; a protein comprising the amino acid substitutions I306A, R130K and G236S; a protein comprising the amino acid substitution R130K; a protein comprising the amino acid substitutions I306A, R130K, G236S and M237W; a protein comprising the amino acid substitutions I306A, G236S and M237W; a protein comprising the amino acid substitutions G236S and M237W; a protein comprising the amino acid substitutions I306A and R130K; a protein comprising the amino acid substitutions R130K and G236S; a protein comprising the amino acid substitutions I306A and F265Y; a protein comprising the amino acid substitutions I306A, G236S and F265Y; a protein comprising the amino acid substitutions R130K, G236S and M237W; a protein comprising the amino acid substitutions G236S and F265Y; a protein comprising the amino acid substitutions R130K, G236S and F265Y; a protein comprising the amino acid substitutions I306A, R130K, G236S and F265Y; a protein comprising the amino acid substitutions I306A, G236S, M237W and F265Y; a protein comprising the amino acid substitution I306A; a protein comprising the amino acid substitution M237W; a protein comprising the amino acid substitution F265Y; a protein comprising the amino acid substitutions I306A and M237W; a protein comprising the amino acid substitutions R130K and M237W; a protein comprising the amino acid substitutions R130K and F265Y; a protein comprising the amino acid substitutions I306A, R130K and M237W; a protein comprising the amino acid substitutions I306A, M237W and F265Y; a protein comprising the amino acid substitutions R130K, M237W and F265Y; a protein comprising the amino acid substitutions G236S, M237W and F265Y; a protein comprising the amino acid substitutions M237W and F265Y; a protein comprising the amino acid substitutions I306A, R130K, M237W and F265Y; a protein comprising the amino acid substitutions R130K, G236S, M237W and F265Y; a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and F265Y, a protein comprising the amino acid substitutions I306A, R130K, G236S and D259A; a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and D259A; a protein comprising the amino acid substitutions I306A, R130K, G236S and N258A; a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and N258A; a protein comprising the amino acid substitutions I306A, R130K, G236S and N258D; a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and N258D; a protein comprising the amino acid substitutions I306A, R130K, G236S and Q193A; a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and Q193A; a protein comprising the amino acid substitutions I306A, R130K, G236S and Q193E; and a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and Q193E; wherein R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1; I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2; G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3; M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3; D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3; Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3.
9. The protein according to claim 1, selected from variant I306A/G236S; variant I306A/R130K/F265Y; variant I306A/R130K/G236S; variant R130K; variant I306A/R130K/G236S/M237W; variant I306A/G236S/M237W; variant G236S/M237W; variant I306A/R130K; variant R130K/G236S; variant I306A/F265Y; variant I306A/G236S/F265Y; variant R130K/G236S/M237W; variant G236S/F265Y; variant R130K/G236S/F265Y; variant I306A/R130K/G236S/F265Y; variant I306A/G236S/M237W/F265Y; variant I306A; variant M237W; variant F265Y; variant I306A/M237W; variant R130K/M237W; variant RI 30K/F265Y; variant I306A/R130K/M237W; variant I306A/M237W/F265Y; variant R130K/M237W/F265Y; variant G236S/M237W/F265Y; variant M237W/F265Y; variant I306A/R130K/M237W/F265Y; variant R130K/G236S/M237W/F265Y; variant I306A/R130K/G236S/M237W/F265Y; variant I306A/RI 30K/G236S/D259A; variant I306A/R130K/G236S/M237W/D259A; variant I306A/R130K/G236S/N258A; variant I306A/R130K/G236S/M237W/N258A; variant I306A/R130K/G236S/N258D; variant I306A/R130K/G236S/M237W/N258D; variant I306A/R130K/G236S/Q193A; variant I306A/R130K/G236S/M237W/Q193A; variant I306A/R130K/G236S/Q193E; and variant I306A/R130K/G236S/M237W/Q193E; wherein R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1; I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2; G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3; M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3; D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3; Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3.
10. The protein according to claim 1, which causes elevated overall production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12) compared to the wild type protein(s) or the protein(s) without such amino acid substitution(s).
11. The protein according to claim 1, wherein the amino acid substitution(s) selected from I306A and G236S; I306A and F265Y; I306A, G236S and M237W; I306A, R130K and G236S; G236S and M237W; I306A, R130K, G236S and M237W; R130K; I306A, G236S and F265Y; I306A, R130K and F265Y; I306A and R130K; R130K and G236S; R130K, G236S and M237W; G236S and F265Y; R130K, G236S and F265Y; I306A, R130K, G236S and F265Y; I306A, G236S, M237W and F265Y; I306A; M237W; F265Y; I306A and M237W; R130K and M237W; R130K and F265Y; I306A, R130K and M237W; I306A, M237W and F265Y; R130K, M237W and F265Y; G236S, M237W and F265Y; M237W and F265Y; I306A, R130K, M237W and F265Y; R130K, G236S, M237W and F265Y; I306A, R130K, G236S, M237W and F265Y; I306A, R130K, G236S and N258A; I306A, R130K, G236S and N258D; I306A, R130K, G236S, M237W and N258A; and I306A, R130K, G236S, M237W and N258D wherein R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1; I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2; G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3; M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3; increase(s) the selectivity for the production of C.sub.6 fatty acids, C.sub.6 fatty acid CoA esters, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, and/or enzyme bound C.sub.6 fatty acids compared to wild type protein(s) or the protein without such amino acid substitution(s).
12. The protein according to claim 1, wherein the amino acid substitution(s) selected from I306A, R130K and F265Y; R130K; I306A, R130K, G236S and M237W; R130K and G236S; I306A and R130K; G236S and M237W; I306A, R130K and G236S; I306A, G236S and M237W; R130K, G236S and M237W; I306A and F265Y; M237W; I306A, G236S and F265Y; G236S and F265Y; I306A and G236S; R130K, G236S and F265Y; I306A, R130K, G236S and F265Y; I306A, G236S, M237W and F265Y; I306A; F265Y; I306A and M237W; R130K and M237W; R130K and F265Y; I306A, R130K and M237W; I306A, M237W and F265Y; R130K, M237W and F265Y; G236S, M237W and F265Y; M237W and F265Y; I306A, R130K, M237W and F265Y; R130K, G236S, M237W and F265Y; I306A, R130K, G236S, M237W and F265Y; I306A, R130K, G236S and D259A, I306A, R130K, G236S and N258A; I306A, R130K, G236S and N258D; I306A, R130K, G236S and Q193A; I306A, R130K, G236S and Q193E; I306A, R130K, G236S, M237W and D259A; I306A, R130K, G236S, M237W and N258A; I306A, R130K, G236S, M237W and N258D; I306A, R130K, G236S, M237W and Q193A; and I306A, R130K, G236S, M237W and Q193E wherein R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1; I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2; G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3; M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3; D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3; Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3; increase(s) the selectivity for the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to wild type protein(s) or the protein without such amino acid substitution(s).
13. The protein according to claim 1, wherein the amino acid substitution(s) selected from I306A, R130K, G236S and M237W; G236S and M237W; R130K; I306A, G236S and F265Y; I306A, R130K and G236S; R130K and G236S; I306A and R130K; I306A, G236S and M237W; I306A and F265Y; M237W; I306A, R130K and F265Y; R130K, G236S and M237W; G236S and F265Y; I306A and G236S; R130K, G236S and F265Y; I306A, R130K, G236S and F265Y; I306A, G236S, M237W and F265Y; I306A; F265Y; I306A and M237W; R130K and M237W; R130K and F265Y; I306A, R130K and M237W; I306A, M237W and F265Y; R130K, M237W and F265Y; G236S, M237W and F265Y; M237W and F265Y; I306A, R130K, M237W and F265Y; R130K, G236S, M237W and F265Y; I306A, R130K, G236S, M237W and F265Y; I306A, R130K, G236S and D259A, I306A, R130K, G236S and N258A; I306A, R130K, G236S and N258D; I306A, R130K, G236S and Q193A; I306A, R130K, G236S and Q193E; I306A, R130K, G236S, M237W and D259A; I306A, R130K, G236S, M237W and N258A; I306A, R130K, G236S, M237W and N258D; I306A, R130K, G236S, M237W and Q193A; and I306A, R130K, G236S, M237W and Q193E wherein R130K refers to R130K in the MPT domain of the amino acid sequence of SEQ ID NO: 1; I306A refers to I306A in the AT domain of the amino acid sequence of SEQ ID NO: 2; G236S refers to G236S in the KS domain of the amino acid sequence of SEQ ID NO: 3; M237W refers to M237W in the KS domain of the amino acid sequence of SEQ ID NO: 3; F265Y refers to F265Y in the KS domain of the amino acid sequence of SEQ ID NO: 3; D259A refers to D259A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258A refers to N258A in the KS domain of the amino acid sequence of SEQ ID NO: 3; N258D refers to N258D in the KS domain of the amino acid sequence of SEQ ID NO: 3; Q193A refers to Q193A in the KS domain of the amino acid sequence of SEQ ID NO: 3; and Q193E refers to Q193E in the KS domain of the amino acid sequence of SEQ ID NO: 3; increase(s) the selectivity for the production of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to wild type protein(s) or the protein without such amino acid substitution(s).
14. A nucleic acid molecule, encoding a a polypeptide domain according to claim 4.
15. A host cell, containing a nucleic acid molecule according to claim 14.
16. The host cell according to claim 15, which has an elevated overall production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12) compared to a cell not containing said nucleic acid molecule, or which has an increased yield of C.sub.6 fatty acids, C.sub.6 fatty acid CoA esters, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, and/or enzyme bound C.sub.6 fatty acids compared to a cell not containing said nucleic acid molecule, or which has an increased yield of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to a cell not containing said nucleic acid molecule, or which has an increased yield of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to a cell not containing said nucleic acid molecule.
17. A method for the production of short fatty acids, CoA esters of short fatty acids, short fatty acid ethyl esters, short fatty acid esters with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12), comprising the expression of a nucleic acid molecule according to claim 14.
18. A method for the production of a biofuel, a flavoring compound and/or a fine chemical (such as natural compounds), comprising the expression of a nucleic acid molecule according to claim 14.
19. A method for: the bulk production of short fatty acids (C.sub.6 to C.sub.12), the specific production of C.sub.6 fatty acids, the specific production of C.sub.8 fatty acids, the bulk production of CoA esters of short fatty acids (C.sub.6 to C.sub.12), the specific production of C.sub.6 fatty acid CoA esters, the specific production of C.sub.8 fatty acid CoA esters, the bulk production of ethyl esters of short fatty acids (C.sub.6 to C.sub.12), the specific production of C.sub.6 fatty acid ethyl esters, the specific production of C.sub.8 fatty acid ethyl esters, or the bulk production of short fatty acids (C.sub.6 to C.sub.12) esters with other metabolites, the specific production of C.sub.6 fatty acid esters with other metabolites, the specific production of C.sub.8 fatty acid esters with other metabolites, the bulk production of enzyme bound short fatty acids (C.sub.6 to C.sub.12), the specific production of enzyme bound C.sub.6 fatty acids, the specific production of enzyme bound C.sub.8 fatty acids, the production of biofuels, the production of fine chemicals, where short fatty acids (C.sub.6 to C.sub.12) or their derivatives are used as a building block, or the production of flavoring substances, wherein said method comprises the use of a nucleic acid molecule according to claim 14.
20. The polypeptide domain, according to claim 4, having at least one amino acid substitution selected from G236, M237, and F265.
21. The host cell, according to claim 15, wherein said host cell is a Saccharomyces cerevisiae.
Description:
CROSS REFERENCE TO A RELATED APPLICATION
[0001] This application is a Divisional Application of co-pending U.S. patent application Ser. No. 15/561,201, filed Sep. 25, 2017 (now U.S. Pat. No. 11,078,468); which is a National Stage Application of International Application Number PCT/EP2016/057167, filed Mar. 31, 2016; which claims priority to European Patent Application No. 15162192.7, filed Apr. 1, 2015; all of which are incorporated herein by reference in their entirety.
[0002] The Sequence Listing for this application is labeled "SeqList-14Sep17-ST25.txt", which was created on Sep. 14, 2017, and is 38 KB. The entire content is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to proteins involved in fatty acid synthesis, such as fatty acid synthases (FAS) variants, comprising one or more polypeptide chains, wherein said polypeptide chain(s) comprise one or more subunits comprising a malonyl/palmitoyl transferase domain (MPT domain), acetyl transferase domain (AT domain), and ketoacyl synthase domain (KS domain), and at least one amino acid substitution in the MPT domain at a position corresponding to R130, in the AT domain at a position corresponding to 1306, and/or in the KS domain, preferably in the acyl binding channel and/or at KS domain binding site to ACP, to modulate affinities of acyl intermediates, and optionally further amino acid substitution(s). The present invention relates to the respective polypeptide domains.
BACKGROUND OF THE INVENTION
[0004] The present invention further relates to nucleic acid molecules encoding the proteins (or the polypeptide domains) and to host cells containing said nucleic acid molecules. The present invention further relates to a method for the production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12), comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention further relates to a method for the production of biofuels, flavoring compounds and/or fine chemicals, comprising the expression of said nucleic acid molecules, preferably in said host cells. The present invention also relates to the use of the proteins, nucleic acids molecule or host cells for the bulk production of short fatty acids (C.sub.6 to C.sub.12), the specific production of C.sub.6 fatty acids and/or C.sub.8 fatty acids, the bulk production of CoA esters of short fatty acids (C.sub.6 to C.sub.12), the specific production of C.sub.6-CoA esters and/or C.sub.8-CoA esters, the bulk production of ethyl esters of short fatty acids (C.sub.6 to C.sub.12), the specific production of C.sub.6 fatty acid ethyl esters and/or C.sub.8 fatty acid ethyl esters, the bulk production of esters of short fatty acids (C.sub.6 to C.sub.12) with other metabolites, the specific production of C.sub.6 fatty acid esters with other metabolites and/or C.sub.8 fatty acid esters with other metabolites, the bulk production of enzyme bound short fatty acids (C.sub.6 to C.sub.12), the specific production of enzyme bound C.sub.6 fatty acids and/or enzyme bound C.sub.8 fatty acids, the production of biofuels, fine chemicals and/or flavoring substances.
[0005] With the rising demand for industrial products from a growing world population, the call for chemicals from renewable sources has become louder. Especially the efforts have been intensified, where fatty acids (FA) can play a crucial role as a platform chemical in the production of fine chemicals or even to replace fossil derived fuel by biofuels (Runguphan & Keasling, 2014; Choi & Lee, 2013). Accordingly, the production of FA in microorganisms has been investigated extensively, constantly pushing the limits: Yields have been shown in S. cerevisiae of up to 400 mg/L free FA (Runguphan & Keasling, 2014) and more recently even 2.2 g/L (Leber et al., 2015) (in both cases products were mostly in the long chain range); and up to 4.8 g/L in E. coli (predominantly C.sub.14 and C.sub.16) (Liu et al., 2012).
[0006] In microorganisms themselves, FA serve several purposes, mainly as a part of membranes, in signaling but also in energy storage. Their de-novo production is tightly regulated and conducted by an enzyme group, the fatty acid synthases (FASs) (Tehlivets et al., 2007). Throughout all organisms, their reaction mechanisms and their chemistry are essentially the same: A ketoacyl synthase (KS) is responsible for the elongation of an acyl chain starter molecule, typically an acetyl-CoA, with malonyl. The resulting .beta.-ketoacyl intermediate is then processed in a series of reaction steps, in the ketoacyl reductase (KR), the dehydratase (DH) and enoyl reductase (ER), to a fully reduced acyl chain. This acyl chain which is now extended by two carbons, serves as a starter for the next cycle. The process repeats itself until the final product is cleft off.
[0007] As far as the overall structural organization of fatty acid synthases (FASs) is concerned, two types are distinguished: In type I FASs, all necessary enzymatic functions of FA production are concentrated in one multienzymatic complex, whereas in type II FAS systems, each reaction is catalyzed by a separate enzyme. Type II FASs are found in bacteria, while type I FASs are typical for few actinobacteria and all eukaryotic organisms, among these also S. cerevisiae.
[0008] In detail, in the S. cerevisiae FAS, one set of domains is distributed on two genes, fas1 and fas2. Multiple copies of the corresponding two polypeptide chains form the heterododecameric .alpha..sub.6.beta..sub.6 2.6 MDa complex, which has been object of extensive x-ray structural analysis with resolutions up to 3.1 .ANG. (see e.g. Jenni et al., 2007; Johansson et al., 2008). Its interpretation has led to substantially new insights in the reaction mechanisms of the whole FAS enzyme family (Grininger 2014; Beld et al., 2015). Above that, the kinetic parameters of S. cerevisiae FAS have been studied for decades.
[0009] The product distribution of the S. cerevisiae FAS is naturally in the long chain range of C.sub.16 and C.sub.18 (Tehlivets et al., 2007) and not directly suitable for applications where short FA are needed, as for instance biofuels in the petrol range. For this purpose, products typically have to have a length of C.sub.4 to C.sub.12 (Peralta-Yahya et al., 2012). Previous engineering efforts for the production of short FA have heavily relied on the utilization of thioesterases (TEs) with known specificities for short chain products (Beld et al., 2015), e.g. in a proof of principle study for the alkane production in E. coli (Choi & Lee, 2013) or the production of the precursors, short FA, in S. cerevisiae (with total yields up to 111 mg/L) (Leber & da Silva, 2014). In contrast, the rational engineering for the production of short FA was believed to be hard to achieve (Beld et al., 2015; Leber & da Silva, 2014).
[0010] US 2003/0145350 A1 discloses DNA sequences which code for a protein having the enzymatic activity of a beta-ketoacyl ACP synthase (KAS) of the enzyme complex of the fatty acid synthase (FAS). US 2003/0145350 A1 further discloses transgenic plants and micro-organisms which containing said nucleic acid sequences and a method for influencing the fatty acid pattern and/or for increasing the fatty acid content, especially the content of short and middle chain fatty acids, in plants, especially in seed tissues that synthesize and/or store triacylglycerols, as well as in micro-organisms, especially bacteria and algae. The method comprises the expression of proteins having the activity of a KAS of the enzyme complex or the fatty acid synthase in transgenic plants or micro-organisms.
[0011] There is a need in the art for further as well as improved means and methods for producing fatty acids in microorganisms, in particular fatty acids that are suitable for biofuel production, fine chemicals and other compounds.
SUMMARY OF THE INVENTION
[0012] According to the present invention this object is solved by a polypeptide or protein involved in fatty acid synthesis, said polypeptide or protein comprising one or more polypeptide chains, wherein said polypeptide chain(s) comprise
(i) one or more subunits comprising the amino acid sequences of
[0013] SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain);
[0014] SEQ ID NO: 2 (acetyl transferase domain, AT domain), and/or
[0015] SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); (ii) at least one amino acid substitution
[0016] in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1;
[0017] in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2;
[0018] and/or
[0019] in the KS domain, preferably in the acyl binding channel, preferably selected from a position corresponding to G236, M237 and F265 of the amino acid sequence of SEQ ID NO: 3; wherein the amino acid sequence comprising the at least one amino acid substitution has at least 60%, or preferably at least 70% or 80% or 90% or 95% sequence identity to the respective amino acid sequence of SEQ ID NO: 1, 2 and/or 3.
[0020] According to the present invention this object is solved by a polypeptide or protein involved in fatty acid synthesis, as defined above, comprising at least one further amino acid substitution in the KS domain, preferably selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3.
[0021] According to the present invention this object is solved by a polypeptide domain comprising
(i) one or more subunits comprising the amino acid sequences of
[0022] SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain);
[0023] SEQ ID NO: 2 (acetyl transferase domain, AT domain), or
[0024] SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); (ii) at least one amino acid substitution
[0025] in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1;
[0026] in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2;
[0027] and/or
[0028] in the KS domain, preferably in the acyl binding channel, preferably selected from a position corresponding to G236, M237 and F265 of the amino acid sequence of SEQ ID NO: 3; wherein the amino acid sequence comprising the at least one amino acid substitution has at least 60%, or preferably at least 70% or 80% or 90% or 95% sequence identity to the respective amino acid sequence of SEQ ID NO: 1, 2 and/or 3.
[0029] According to the present invention this object is solved by a polypeptide domain, as defined above, comprising at least one further amino acid substitution in the KS domain, preferably selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3.
[0030] According to the present invention this object is solved by a nucleic acid molecule coding for a protein of the present invention or a polypeptide domain of the present invention.
[0031] According to the present invention this object is solved by a host cell, containing a nucleic acid molecule of the present invention and preferably expressing said nucleic acid molecule, wherein said host cell is preferably selected from a bacterial cell or a fungus cell, more preferably a yeast cell, or an algae cell.
[0032] According to the present invention this object is solved by a method for the production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12), comprising the expression of a nucleic acid molecule according to the present invention, preferably in a host cell according to the present invention.
[0033] According to the present invention this object is solved by a method for the production of biofuels, flavoring compounds and/or fine chemicals comprising the expression of a nucleic acid molecule according to the present invention, preferably in a host cell according to the present invention.
[0034] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the bulk production of short fatty acids (C.sub.6 to C.sub.12).
[0035] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of C.sub.6 fatty acids.
[0036] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of C.sub.8 fatty acids.
[0037] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the bulk production of CoA esters of short fatty acids (C.sub.6 to C.sub.12).
[0038] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of C.sub.6-CoA esters.
[0039] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of C.sub.8-CoA esters.
[0040] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the bulk production of ethyl esters of short fatty acids (C.sub.6 to C.sub.12) or esters of short fatty acids (C.sub.6 to C.sub.12) with other metabolites.
[0041] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of C.sub.6 fatty acid ethyl esters or C.sub.6 fatty acid esters with other metabolites.
[0042] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of C.sub.8 fatty acid ethyl esters or C.sub.8 fatty acid esters with other metabolites.
[0043] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the bulk production of enzyme bound short fatty acids (C.sub.6 to C.sub.12).
[0044] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of enzyme bound C.sub.6 fatty acids.
[0045] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the specific production of enzyme bound C.sub.8 fatty acids.
[0046] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the production of biofuels, such as short alkanes, short alkenes, short alkynes, short esters and/or alcohols.
[0047] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the production of fine chemicals, such as natural compounds where preferably short fatty acids (C.sub.6 to C.sub.12) or their derivatives (such as CoA esters, methyl/ethyl esters, esters with other metabolites, enzyme bound fatty acids, alcohols) are used as building block(s).
[0048] According to the present invention this object is solved by using a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention for the production of flavoring substances, such as esters from short fatty acids (C.sub.6 to C.sub.12).
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[0049] Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.
Fatty Acid Synthase Variants and Domains
[0050] As discussed above, the present invention provides fatty acid synthase (FAS) variants, in particular type I fatty acid synthase (type I FAS) variants.
[0051] The polypeptides or proteins of the present invention comprise one or more polypeptide chains. Said one or more polypeptide chains comprise one or more subunits comprising a malonyl/palmitoyl transferase domain (MPT domain), an acetyl transferase domain (AT domain), and/or a ketoacyl synthase domain (KS domain).
[0052] The invention provides a protein or polypeptide involved in fatty acid synthesis, preferably having fatty acid synthase activity.
[0053] The invention also provides polypeptide domain(s) which are involved in fatty acid synthesis activity, such as subunits comprising a malonyl/palmitoyl transferase domain (MPT domain), an acetyl transferase domain (AT domain), and/or a ketoacyl synthase domain (KS domain).
[0054] Fatty acid synthases or polypeptides/proteins involved in fatty acid synthesis comprise one or more polypeptide chains, such as two, three, four or more polypeptide chains.
[0055] Fatty acid synthases or polypeptides/proteins involved in fatty acid synthesis furthermore comprise several different catalytic domains or subunits. Said catalytic domains or subunits can be located on said different/one or more polypeptide chains. The catalytic domains or subunits can also be split in parts and the different parts can be located on said different/one or more polypeptide chains (such as in case for the MPT domain of the type I FAS from S. cerevisiae).
[0056] According to the invention, the polypeptides or proteins of the present invention further comprise at least one amino acid substitution in the MPT domain at a position corresponding to R130, in the AT domain at a position corresponding to 1306, and/or in the KS domain, preferably in the acyl binding channel, to modulate affinities of acyl intermediates, and optionally further amino acid substitution(s). In particular, the present invention provides a polypeptide or protein involved in fatty acid synthesis, said polypeptide comprising one or more polypeptide chains, said polypeptide chain(s) comprising
(i) at least one subunit comprising the amino acid sequences of
[0057] SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain);
[0058] SEQ ID NO: 2 (acetyl transferase domain, AT domain), and/or
[0059] SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); (ii) at least one amino acid substitution
[0060] in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1;
[0061] in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2;
[0062] and/or
[0063] in the KS domain, preferably in the acyl binding channel, preferably selected from a position corresponding to G236, M237 and F265 of the amino acid sequence of SEQ ID NO: 3.
[0064] In particular, the present invention provides a polypeptide or protein involved in fatty acid synthesis, as defined above, comprising at least one further amino acid substitution in the KS domain, preferably in the acyl binding channel and/or at KS domain binding site to ACP, preferably selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3.
[0065] According to the present invention, the amino acid sequence comprising the at least one amino acid substitution has at least 60%, or preferably at least 70% or 80% or 90% or 95% sequence identity to the respective amino acid sequence of SEQ ID NO: 1, 2 and/or 3.
[0066] In a preferred embodiment, the amino acid sequence comprising the at least one amino acid substitution has at least 80%, or preferably at least 90% or 95% sequence identity to the respective amino acid sequence of SEQ ID NO: 3.
[0067] In particular, the present invention provides a polypeptide domain comprising
(i) one or more subunits comprising the amino acid sequences of
[0068] SEQ ID NO: 1 (malonyl/palmitoyl transferase domain, MPT domain);
[0069] SEQ ID NO: 2 (acetyl transferase domain, AT domain), or
[0070] SEQ ID NO: 3 (ketoacyl synthase domain, KS domain); (ii) at least one amino acid substitution
[0071] in the MPT domain at a position corresponding to R130 of the amino acid sequence of SEQ ID NO: 1;
[0072] in the AT domain at a position corresponding to 1306 of the amino acid sequence of SEQ ID NO: 2;
[0073] and/or
[0074] in the KS domain, preferably in the acyl binding channel, preferably selected from a position corresponding to G236, M237 and F265 of the amino acid sequence of SEQ ID NO: 3;
[0075] In particular, the present invention provides a polypeptide domain, as defined above, comprising at least one further amino acid substitution in the KS domain, preferably at KS domain binding site to ACP, preferably selected from a position corresponding to Q193, N258 and D259 of the amino acid sequence of SEQ ID NO: 3.
[0076] According to the present invention, the amino acid sequence comprising the at least one amino acid substitution has at least 60%, or preferably at least 70% or 80% or 90% or 95% sequence identity to the respective amino acid sequence of SEQ ID NO: 1, 2 and/or 3.
[0077] In a preferred embodiment, the amino acid sequence comprising the at least one amino acid substitution has at least 80%, or preferably at least 90% or 95% sequence identity to the respective amino acid sequence of SEQ ID NO: 3.
[0078] In a preferred embodiment, the MPT and/or AT domain of the polypeptide(s) or protein(s) (or the polypeptide domain(s)) have an in vitro and/or in vivo transferase activity; and/or the KS domain of the polypeptide(s) or protein(s) has an in vitro and/or in vivo ketoacyl synthase activity.
[0079] This means that even though there is at least one amino acid substitution in the MPT domain, AT domain and/or KS domain, as disclosed herein, the MPT and/or AT domain of the polypeptide(s) or protein(s) have an in vitro and/or in vivo transferase activity; and/or the KS domain of the polypeptide(s) or protein(s) has an in vitro and/or in vivo ketoacyl synthase activity.
[0080] There are two principal classes of fatty acid synthases.
[0081] Type I systems utilize a single large, multifunctional enzyme and are common to both mammals and fungi (although the structural arrangement of fungal and mammalian synthases differ). A Type 1 fatty acid synthase system is also found in the CMN group of bacteria (corynebacteria, mycobacteria, and nocardia). In these bacteria, the FAS I system produces palmititic acid, and cooperates with the FAS II system to produce a greater diversity of lipid products. Type II is found in archaea and bacteria, and is characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis.
[0082] For example, mammalian FAS usually consists of a homodimer of two identical protein subunits, in which three catalytic domains in the N-terminal section (-ketoacyl synthase (KS), malonyl/acetyltransferase (MAT), and dehydratase (DH)), are separated by a core region of 600 residues from four C-terminal domains (enoyl reductase (ER), -ketoacyl reductase (KR), acyl carrier protein (ACP) and thioesterase (TE)).
[0083] In one embodiment, the protein or polypeptide of the present invention is a type I FAS, such as type I FAS of Saccharomyces cerevisiae. In the case of Saccharomyces cerevisiae, the FAS is split into two polypeptide chains, but still is considered a type I FAS since the catalytic centers are organized in one enzyme complex.
[0084] In one embodiment, the protein or polypeptide of the present invention is the type I FAS of Saccharomyces cerevisiae.
[0085] The particular type I FAS from S. cerevisiae comprises two polypeptide chains which among other domains, contain
[0086] on a first polypeptide chain FAS 1:
[0087] the malonyl/palmitoyl transferase domain, MPT domain
[0088] and
[0089] the acetyl transferase domain, AT domain
[0090] on a second polypeptide chain, FAS 2
[0091] ketoacyl synthase domain, KS domain.
[0092] In S. cerevisiae, the chains are encoded by fas1 (to be found in Genbank: M30162.1) and fas2 (to be found in Genbank: J03936.1).
[0093] SEQ ID NO: 1 shows the amino acid sequence of the MPT domain (part of sequence of Uniprot Identifier: P07149 and of P19097)
[0094] Malonyl/palmitoyl transferase (MPT domain), (sequence is split in S. cerevisiae with the splitting site indicated by *: from residue 1-347 shown here, it is part of S. cerevisiae FAS 1 (beta chain), Uniprot Identifier: P07149; from residue 348-429 shown here, it is part of S. cerevisiae FAS 2 (beta alpha), Uniprot Identifier: P19097)
TABLE-US-00001 SILDIVINNPVNLTIHFGGEKGKRIRENYSAMIFETIVDGKLKTEKIFK EINEHSTSYTFRSEKGLLSATQFTQPALTLMEKAAFEDLKSKGLIPADA TFAGHSLGEYAALASLADVMSIESLVEVVFYRGMTMQVAVPRDELGRSN YGMIAINPGRVAASFSQEALQYVVERVGKRTGWLVEIVNYNVENQQYVA AGDLRALDTVTNVLNFIKLQKIDIIELQKSLSLEEVEGHLFEIIDEASK KSAVKPRPLKLERGFACIPLVGISVPFHSTYLMNGVKPFKSFLKKNIIK ENVKVARLAGKYIPNLTAKPFQVTKEYFQDVYDLTGSEPIKEIIDN WEKYEQS * MKPEVEQELAHILLTELLAYQFASPVRWIETQDVFLKDFNTERVVEIGP SPTLAGMAQRTLKNKYESYDAALSLHREILCYS
[0095] SEQ ID NO: 2 shows the amino acid sequence of the AT domain (part of sequence of Uniprot Identifier: P07149)
Acetyl transferase (AT domain), (part of S. cerevisiae FAS 1 (beta chain),
Uniprot Identifier: P07149
TABLE-US-00002
[0096] MDAYSTRPLTLSHGSLEHVLLVPTASFFIASQLQEQFNKILPEPTEGFA ADDEPTTPAELVGKFLGYVSSLVEPSKVGQFDQVLNLCLTEFENCYLEG NDIHALAAKLLQENDTTLVKTKELIKNYITARIMAKRPFDKKSNSALFR AVGEGNAQLVAIFGGQGNTDDYFEELRDLYQTYHVLVGDLIKFSAETLS ELIRTTLDAEKVFTQGLNILEWLENPSNTPDKDYLLSIPISCPLIGVIQ LAHYVVTAKLLGFTPGELRSYLKGATGHSQGLVTAVAIAETDSWESFFV SVRKAITVLFFIGVRCYEAYPNTSLPPSILEDSLENNEGVPSPMLSISN LTQEQVQDYVNKTNSHLPAGKQVEISLVNGAKNLVVSGPPQSLYGLNLT LRKAKAPSGLDQSRIPFSERKLKFSNRFLPVASPFHSHLLVPASDLINK DLVKNNVSFNAKDIQIPVYDTFDGSDLRVLSGSISERIVDCIIRLPVKW ETTTQFKATHILDFGPGGASGLGVLTHRNKDGTGVRVIVAGTLDINPDD DYGFKQEIFDVT
[0097] SEQ ID NO: 3 shows the amino acid sequence of the KS domain (part of sequence of Uniprot Identifier: P19097)
[0098] Ketoacyl synthase (KS domain), (part of S. cerevisiae FAS 2 (alpha chain), Uniprot Identifier: P19097)
TABLE-US-00003 LERVIVVTGFAEVGPWGSARTRWEMEAFGEFSLEGCVEMAWIMGFISYH NGNLKGRPYTGWVDSKTKEPVDDKDVKAKYETSILEHSGIRLIEPELFN GYNPEKKEMIQEVIVEEDLEPFEASKETAEQFKHQHGDKVDIFEIPETG EYSVKLLKGATLYIPKALRFDRLVAGQIPTGWNAKTYGISDDIISQVDP ITLFVLVSVVEAFIASGITDPYEMYKYVHVSEVGNCSGSGMGGVSALRG MFKDRFKDEPVQNDILQESFINTMSAWVNMLLISSSGPIKTPVGACATS VESVDIGVETILSGKARICIVGGYDDFQEEGSFEFGNMKATSNTLEEFE HGRTPAEMSRPATTTRNGFMEAQGAGIQIIMQADLALKMGVPIYGIVAM AATATDKIGRSVPAPGKGILTTAREHHSSVKYASPNLNMKYRKRQLVTR EAQIKDWVENELEALKLEAEEIPSEDQNEFLLERTREIHNEAESQLRAA QQQWGNDFYKRDPRIAPLRGALATYGLTIDDLGVASFHGTSTKANDKNE SATINEMMKHLGRSEGNPVIGVFQKFLTGHPKGAAGAWMMNGALQILNS GIIPGNRNADNVDKILEQFEYVLYPSKTLKTDGVRAVSITSFGFGQKGG QAIVVHPDYLYGA
[0099] As far as the overall structural organization of fatty acid synthases (FASs) is concerned, two types are distinguished: In type I FASs, all necessary enzymatic functions of fatty acid (FA) production are concentrated in one multienzymatic complex, whereas in type II FAS systems, each reaction is catalyzed by a separate enzyme. For type I FAS systems, the intermediates are always covalently bound to the multienzymatic complex leading to extremely high efficiency. Type II FASs are found in bacteria, while type I FASB are typical for few actinobacteria and all eukaryotic organisms, among these also S. cerevisiae.
[0100] In detail, in the S. cerevisiae FAS, one set of domains is distributed on two genes, fas1 (encoding the .beta. chain or FAS1) and fas2 (encoding the .alpha. chain or FAS2). On the 13 chain, the AT domain, the ER domain, the DH domain and part of the MPT domain are located in the order as written here. The rest of the MPT domain, the ACP domain, the KR domain, the KS domain and the PPT domain can be found on the .alpha. chain correspondingly. Multiple copies of the corresponding two polypeptide chains form the heterododecameric .alpha..sub.6.beta..sub.6 2.6 MDa complex, which has been object of extensive x-ray structural analysis with resolutions up to 3.1 .ANG. (see e.g. Jenni et al., 2007; Johansson et al., 2008). Its interpretation has led to substantially new insights in the reaction mechanisms of the whole FAS enzyme family (Grininger 2014; Beld et al., 2015). Above that, the kinetic parameters of S. cerevisiae FAS have been studied for decades.
[0101] As used herein, the term "at a position corresponding to" means the respective position in SEQ ID No: 1, 2 or 3 which, however, in related polypeptide chains can have another relative position number. The equivalent substitution can be determined by comparing a position in both sequences, which may be aligned for the purpose of comparison. The relative position of the amino acid can vary due to different length of the related polypeptide, or deletions or additions of amino acids in the related polypeptide.
[0102] The polypeptides of the present invention, in particular the type I FAS variants, have an in vitro and/or in vivo fatty acid synthase I (FAS I) enzymatic activity.
[0103] As used herein, the term "percent (%) identical" refers to sequence identity between two amino acid sequences. Identity can be determined by comparing a position in both sequences, which may be aligned for the purpose of comparison. When an equivalent position in the compared sequences is occupied by the same amino acid, the molecules are considered to be identical at that position.
[0104] As used herein, the term "functional equivalent" refers to amino acid sequences that are not 100% identical to the amino acid sequence of SEQ ID NO. 1, 2 or 3 and comprise amino acid additions and/or insertions and/or deletions and/or substitutions and/or exchanges, which do not alter or change the activity or function of the protein as compared to the protein having the amino acid sequence of SEQ ID NO: 1, 2 or 3, i.e. an "functional equivalent", for example, encompasses an amino acid sequence with conservative amino acid substitutions or smaller deletions and/or insertions as long as these modifications do not substantially affect the in vitro and/or in vivo fatty acid synthase (FAS) enzymatic activity.
[0105] Generally, a person skilled in the art is aware of the fact that some amino acid exchanges in the amino acid sequence of a protein do not have an influence on the (secondary or tertiary) structure, function and/or activity of that protein. Amino acid sequences with such "neutral" amino acid exchanges as compared to the amino acid sequences disclosed herein fall within the scope of the present invention.
[0106] In some embodiments, the polypeptide(s) or protein(s) have one, two, three, four, five, six or more amino acid substitutions in the MPT domain, AT domain and/or KS domain.
[0107] For example, a polypeptide of the present inventions has one amino acid substitution in each of the MPT domain, AT domain and/or KS domain.
[0108] For example, a polypeptide of the present inventions has two amino acid substitutions, such as:
one amino acid substitution in each of the MPT domain and the AT domain, one amino acid substitution in each of the MPT domain and the KS domain, one amino acid substitution in each of the AT domain and the KS domain, two amino acid substitutions in the MPT domain, two amino acid substitutions in the AT domain, two amino acid substitutions in the KS domain.
[0109] For example, a polypeptide of the present inventions has three amino acid substitutions, such as:
three amino acid substitutions in the MPT domain, three amino acid substitutions in the AT domain, three amino acid substitutions in the KS domain. two amino acid substitutions in the MPT domain and one amino acid substitution in either one of the AT domain and the KS domain, two amino acid substitutions in the AT domain and one amino acid substitution in either one of the MPT domain and the KS domain, two amino acid substitutions in the KS domain and one amino acid substitution in either one of MPT domain and the AT domain, one amino acid substitution in each of the MPT domain, AT domain and KS domain.
[0110] Preferably, the polypeptides or proteins according to the present invention comprise
[0111] the amino acid substitution R130K in the MPT domain (SEQ ID NO. 1), and/or
[0112] the amino acid substitution I306A in the AT domain (SEQ ID NO: 2).
[0113] Preferably, the polypeptides or proteins according to the present invention comprise amino acid substitution(s) in the acyl binding channel of the KS domain, more preferably amino acid substitution(s) G236S, M237W and/or F265Y in the KS domain (SEQ ID NO: 3).
[0114] Preferably, the polypeptides or proteins according to the present invention comprise
[0115] the amino acid substitution R130K in the MPT domain (SEQ ID NO. 1), and/or
[0116] the amino acid substitution I306A in the AT domain (SEQ ID NO: 2), and/or
[0117] the amino acid substitution(s) G236S, M237W and/or F265Y in the KS domain (SEQ ID NO: 3) and/or
[0118] the amino acid substitution(s) Q193A, Q193E, N258A, N258D and/or D259A in the KS domain (SEQ ID NO: 3).
Disclaimer:
[0119] The present invention does not encompass polypeptide domain(s) that only comprise the amino acid substitution G236S in the KS domain.
[0120] In an embodiment, where the polypeptide domain of the present invention comprises the KS domain and an amino acid substitution G236S in said KS domain (SEQ ID NO. 3), it comprises at least one additional amino acid substitution in said KS domain.
[0121] The present invention does not encompass polypeptides or proteins that only comprise the amino acid substitution G236S in the KS domain.
[0122] In an embodiment, where the polypeptide or protein of the present invention comprises the amino acid substitution G236S in the KS domain (SEQ ID NO. 3), it comprises at least one additional amino acid substitution.
[0123] Preferably, the proteins or polypeptides according to the present invention are selected from the group of
[0124] a protein comprising the amino acid substitutions I306A and G236S;
[0125] a protein comprising the amino acid substitutions I306A, R130K and F265Y;
[0126] a protein comprising the amino acid substitutions I306A, R130K and G236S;
[0127] a protein comprising the amino acid substitution R130K;
[0128] a protein comprising the amino acid substitutions I306A, R130K, G236S and M237W;
[0129] a protein comprising the amino acid substitutions I306A, G236S and M237W;
[0130] a protein comprising the amino acid substitutions G236S and M237W;
[0131] a protein comprising the amino acid substitutions I306A and R130K;
[0132] a protein comprising the amino acid substitutions R130K and G236S;
[0133] a protein comprising the amino acid substitutions I306A and F265Y;
[0134] a protein comprising the amino acid substitutions I306A, G236S and F265Y;
[0135] a protein comprising the amino acid substitutions R130K, G236S and M237W;
[0136] a protein comprising the amino acid substitutions G236S and F265Y;
[0137] a protein comprising the amino acid substitutions R130K, G236S and F265Y;
[0138] a protein comprising the amino acid substitutions I306A, R130K, G236S and F265Y;
[0139] a protein comprising the amino acid substitutions I306A, G236S, M237W and F265Y;
[0140] a protein comprising the amino acid substitution I306A;
[0141] a protein comprising the amino acid substitution M237W;
[0142] a protein comprising the amino acid substitution F265Y;
[0143] a protein comprising the amino acid substitutions I306A and M237W;
[0144] a protein comprising the amino acid substitutions R130K and M237W;
[0145] a protein comprising the amino acid substitutions R130K and F265Y;
[0146] a protein comprising the amino acid substitutions I306A, R130K and M237W;
[0147] a protein comprising the amino acid substitutions I306A, M237W and F265Y;
[0148] a protein comprising the amino acid substitutions R130K, M237W and F265Y;
[0149] a protein comprising the amino acid substitutions G236S, M237W and F265Y;
[0150] a protein comprising the amino acid substitutions M237W and F265Y;
[0151] a protein comprising the amino acid substitutions I306A, R130K, M237W and F265Y;
[0152] a protein comprising the amino acid substitutions R130K, G236S, M237W and F265Y;
[0153] a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and F265Y;
[0154] a protein comprising the amino acid substitutions I306A, R130K, G236S and D259A;
[0155] a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and D259A;
[0156] a protein comprising the amino acid substitutions I306A, R130K, G236S and N258A;
[0157] a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and N258A;
[0158] a protein comprising the amino acid substitutions I306A, R130K, G236S and N258D;
[0159] a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and N258D;
[0160] a protein comprising the amino acid substitutions I306A, R130K, G236S and Q193A;
[0161] a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and Q193A;
[0162] a protein comprising the amino acid substitutions I306A, R130K, G236S and Q193E;
[0163] a protein comprising the amino acid substitutions I306A, R130K, G236S, M237W and Q193E.
[0164] The present invention preferably provides the following proteins or polypeptides/type I FAS variants:
[0165] variant I306A/G236S;
[0166] variant I306A/R130K/F265Y;
[0167] variant I306A/R130K/G236S;
[0168] variant R130K;
[0169] variant I306A/R130K/G236S/M237W;
[0170] variant I306A/G236S/M237W;
[0171] variant G236S/M237W;
[0172] variant I306A/R130K;
[0173] variant R130K/G236S;
[0174] variant I306A/F265Y;
[0175] variant I306A/G236S/F265Y;
[0176] variant R130K/G236S/M237W;
[0177] variant G236S/F265Y;
[0178] variant R130K/G236S/F265Y;
[0179] variant I306A/R130K/G236S/F265Y;
[0180] variant I306A/G236S/M237W/F265Y;
[0181] variant I306A;
[0182] variant M237W;
[0183] variant F265Y;
[0184] variant I306A/M237W;
[0185] variant R130K/M237W;
[0186] variant R130K/F265Y;
[0187] variant I306A/R130K/M237W;
[0188] variant I306A/M237W/F265Y;
[0189] variant R130K/M237W/F265Y;
[0190] variant G236S/M237W/F265Y;
[0191] variant M237W/F265Y;
[0192] variant I306A/R130K/M237W/F265Y;
[0193] variant R130K/G236S/M237W/F265Y;
[0194] variant I306A/R130K/G236S/M237W/F265Y;
[0195] variant I306A/R130K/G236S/D259A,
[0196] variant I306A/R130K/G236S/M237W/D259A;
[0197] variant I306A/R130K/G236S/N258A;
[0198] variant I306A/R130K/G236S/M237W/N258A;
[0199] variant I306A/R130K/G236S/N258D;
[0200] variant I306A/R130K/G236S/M237W/N258D;
[0201] variant I306A/R130K/G236S/Q193A;
[0202] variant I306A/R130K/G236S/M237W/Q193A;
[0203] variant I306A/R130K/G236S/Q193E; and
[0204] variant I306A/R130K/G236S/M237W/Q193E.
[0205] Preferably, the protein (s) or polypeptide(s) of the present invention result in elevated overall production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12) compared to the wild type polypeptide(s) or the polypeptide(s) without such amino acid substitution(s).
[0206] The elevated overall production of short fatty acids, CoA esters of short fatty acids, short fatty acid ethyl esters, short fatty acid esters with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12) is increased at least 2-fold, or preferably at least 5-fold or 10-fold or 20-fold or 27-fold.
[0207] The term "short fatty acid" refers to a fatty acid of short to medium length with C.sub.6 to C.sub.12.
[0208] Variants with Increased Selectivity for C6
[0209] In a preferred embodiment, the polypeptide(s) of the present invention show(s) an increased selectivity for the production of C.sub.6 fatty acids, C.sub.6 CoA esters, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, and/or enzyme bound C.sub.6 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0210] The selectivity for the production of C.sub.6 fatty acids, C.sub.6 CoA esters, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, and/or enzyme bound C.sub.6 fatty acids compared to wild type is increased where the share of C.sub.6 makes up at least 30%, or preferably at least 40% or 50% or 70% or 80% or 90% of the detected chain length between C.sub.6 and C.sub.12.
[0211] In particular, proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0212] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0213] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0214] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0215] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0216] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0217] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0218] R130K (in the MPT domain on SEQ ID NO: 1); I306A (in the AT domain on SEQ ID NO: 2), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0219] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0220] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: 1);
[0221] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0222] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0223] G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0224] R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0225] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0226] I306A (in the AT domain on SEQ ID NO: 2), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0227] I306A (in the AT domain on SEQ ID NO: 2);
[0228] M237W (in the KS domain on SEQ ID NO: 3);
[0229] F265Y (in the KS domain on SEQ ID NO: 3);
[0230] I306A (in the AT domain on SEQ ID NO: 2) and M237W (in the KS domain on SEQ ID NO: 3);
[0231] R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0232] R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0233] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0234] I306A (in the AT domain on SEQ ID NO: 2), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0235] R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0236] G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0237] M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0238] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0239] R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0240] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0241] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258A (in the KS domain on SEQ ID NO: 3);
[0242] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258D (in the KS domain on SEQ ID NO: 3);
[0243] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258A (in the KS domain on SEQ ID NO: 3);
[0244] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258D (in the KS domain on SEQ ID NO: 3); preferably selected from
[0245] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0246] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0247] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0248] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3)
[0249] G236S and M237W (in the KS domain on SEQ ID NO: 3). increase(s) the selectivity for the production of C.sub.6 fatty acids, C.sub.6 CoA, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, esters and/or enzyme bound C.sub.6 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0250] Namely, proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0251] I306A and G236S;
[0252] I306A and F265Y;
[0253] I306A, G236S and M237W;
[0254] I306A, R130K and G236S;
[0255] G236S and M237W;
[0256] I306A, R130K, G236S and M237W;
[0257] R130K;
[0258] I306A, G236S and F265Y;
[0259] I306A, R130K and F265Y;
[0260] I306A and R130K;
[0261] R130K and G236S;
[0262] R130K, G236S and M237W;
[0263] G236S and F265Y;
[0264] R130K, G236S and F265Y;
[0265] I306A, R130K, G236S and F265Y;
[0266] I306A, G236S, M237W and F265Y;
[0267] I306A;
[0268] M237W;
[0269] F265Y;
[0270] I306A and M237W;
[0271] R130K and M237W;
[0272] R130K and F265Y;
[0273] I306A, R130K and M237W;
[0274] I306A, M237W and F265Y;
[0275] R130K, M237W and F265Y;
[0276] G236S, M237W and F265Y;
[0277] M237W and F265Y;
[0278] I306A, R130K, M237W and F265Y;
[0279] R130K, G236S, M237W and F265Y;
[0280] I306A, R130K, G236S, M237W and F265Y;
[0281] I306A, R130K, G236S and N258A;
[0282] I306A, R130K, G236S and N258D;
[0283] I306A, R130K, G236S, M237W and N258A;
[0284] I306A, R130K, G236S, M237W and N258D; preferably selected from
[0285] I306A and G236S;
[0286] I306A and F265Y;
[0287] I306A, G236S and M237W;
[0288] I306A, R130K and G236S;
[0289] G236S and M237W; more preferably
[0290] I306A and G236S,
[0291] I306A and F265Y,
[0292] I306A, G236S and M237W,
[0293] I306A, R130K and G236S, increase(s) the selectivity for the production of C.sub.6 fatty acids, C.sub.6 fatty acid CoA esters, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, esters and/or enzyme bound C.sub.6 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0294] Variants with Increased Selectivity for C8
[0295] In a preferred embodiment, the protein (s) or polypeptide(s) of the present invention show(s) an increased selectivity for the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0296] The selectivity for the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters and/or enzyme bound C.sub.8 fatty acids compared to wild type is increased where the share of C.sub.8 makes up at least 30%, or preferably at least 40% or 50% or 70% or 80% or 89% of the detected chain length between C.sub.6 and C.sub.12.
[0297] In particular, proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0298] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3)
[0299] R130K (in the MPT domain on SEQ ID NO: 1);
[0300] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0301] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0302] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: 1);
[0303] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0304] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0305] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0306] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0307] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0308] M237W (in the KS domain on SEQ ID NO: 3);
[0309] I306A (in the AT domain on SEQ ID NO: 2), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0310] G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0311] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0312] R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3)
[0313] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0314] I306A (in the AT domain on SEQ ID NO: 2), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0315] I306A (in the AT domain on SEQ ID NO: 2);
[0316] F265Y (in the KS domain on SEQ ID NO: 3);
[0317] I306A (in the AT domain on SEQ ID NO: 2) and M237W (in the KS domain on SEQ ID NO: 3);
[0318] R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0319] R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0320] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0321] I306A (in the AT domain on SEQ ID NO: 2), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0322] R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0323] G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3); M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0324] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0325] R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0326] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0327] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and D259A (in the KS domain on SEQ ID NO: 3);
[0328] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and D259A (in the KS domain on SEQ ID NO: 3);
[0329] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258A (in the KS domain on SEQ ID NO: 3);
[0330] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258A (in the KS domain on SEQ ID NO: 3);
[0331] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258D (in the KS domain on SEQ ID NO: 3);
[0332] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258D (in the KS domain on SEQ ID NO: 3);
[0333] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0334] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0335] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193E (in the KS domain on SEQ ID NO: 3);
[0336] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193E (in the KS domain on SEQ ID NO: 3); preferably selected from
[0337] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3)
[0338] R130K (in the MPT domain on SEQ ID NO: 1);
[0339] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0340] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0341] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: 1);
[0342] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0343] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0344] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0345] R130K (in the MPT domain on SEQ ID NO: I), G236S and M237W (in the KS domain on SEQ ID NO: 3); increase(s) the selectivity for the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0346] Namely, proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0347] I306A, R130K and F265Y;
[0348] R130K;
[0349] I306A, R130K, G236S and M237W;
[0350] R130K and G236S;
[0351] I306A and R130K;
[0352] G236S and M237W;
[0353] I306A, R130K and G236S;
[0354] I306A, G236S and M237W;
[0355] R130K, G236S and M237W;
[0356] I306A and F265Y;
[0357] M237W;
[0358] I306A, G236S and F265Y;
[0359] G236S and F265Y;
[0360] I306A and G236S;
[0361] R130K, G236S and F265Y;
[0362] I306A, R130K, G236S and F265Y;
[0363] I306A, G236S, M237W and F265Y;
[0364] I306A;
[0365] F265Y;
[0366] I306A and M237W;
[0367] R130K and M237W;
[0368] R130K and F265Y;
[0369] I306A, R130K and M237W;
[0370] I306A, M237W and F265Y;
[0371] R130K, M237W and F265Y;
[0372] G236S, M237W and F265Y;
[0373] M237W and F265Y;
[0374] I306A, R130K, M237W and F265Y;
[0375] R130K, G236S, M237W and F265Y;
[0376] I306A, R130K, G236S, M237W and F265Y;
[0377] I306A, R130K, G236S and D259A,
[0378] I306A, R130K, G236S and N258A;
[0379] I306A, R130K, G236S and N258D;
[0380] I306A, R130K, G236S and Q193A;
[0381] I306A, R130K, G236S and Q193E;
[0382] I306A, R130K, G236S, M237W and D259A;
[0383] I306A, R130K, G236S, M237W and N258A;
[0384] I306A, R130K, G236S, M237W and N258D;
[0385] I306A, R130K, G236S, M237W and Q193A; and
[0386] I306A, R130K, G236S, M237W and Q193E; preferably selected from
[0387] I306A, R130K and F265Y;
[0388] R130K;
[0389] I306A, R130K, G236S and M237W;
[0390] R130K and G236S;
[0391] I306A and R130K;
[0392] G236S and M237W
[0393] I306A, R130K and G236S;
[0394] I306A, G236S and M237W;
[0395] R130K, G236S and M237W; more preferably selected from
[0396] I306A, R130K and F265Y;
[0397] R130K;
[0398] I306A, R130K, G236S and M237W;
[0399] R130K and G236S;
[0400] I306A and R130K;
[0401] G236S and M237W;
[0402] I306A, R130K and G236S; increase(s) the selectivity for the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0403] Variants with Increased Selectivity for C10 to C12
[0404] In a preferred embodiment, the protein (s) or polypeptide(s) of the present invention show(s) an increased selectivity for the production of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0405] In particular, proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0406] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0407] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0408] R130K (in the MPT domain on SEQ ID NO: 1);
[0409] I306A (in the AT domain on SEQ ID NO: 2), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0410] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0411] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0412] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: I);
[0413] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0414] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0415] M237W (in the KS domain on SEQ ID NO: 3);
[0416] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0417] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0418] G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0419] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0420] R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0421] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0422] I306A (in the AT domain on SEQ ID NO: 2), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0423] I306A (in the AT domain on SEQ ID NO: 2);
[0424] F265Y (in the KS domain on SEQ ID NO: 3);
[0425] I306A (in the AT domain on SEQ ID NO: 2) and M237W (in the KS domain on SEQ ID NO: 3);
[0426] R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0427] R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0428] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0429] I306A (in the AT domain on SEQ ID NO: 2), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0430] R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0431] G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0432] M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0433] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0434] R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0435] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0436] I306A (in the AT domain on SEQ ID NO: 2), RI 30K (in the MPT domain on SEQ ID NO: 1), G236S and D259A (in the KS domain on SEQ ID NO: 3);
[0437] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and D259A (in the KS domain on SEQ ID NO: 3);
[0438] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258A (in the KS domain on SEQ ID NO: 3);
[0439] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258A (in the KS domain on SEQ ID NO: 3);
[0440] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258D (in the KS domain on SEQ ID NO: 3);
[0441] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258D (in the KS domain on SEQ ID NO: 3);
[0442] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0443] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0444] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193E (in the KS domain on SEQ ID NO: 3);
[0445] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193E (in the KS domain on SEQ ID NO: 3); preferably selected from
[0446] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0447] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0448] R130K (in the MPT domain on SEQ ID NO: 1); increase(s) the selectivity for the production of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0449] Namely, proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0450] I306A, R130K, G236S and M237W;
[0451] G236S and M237W;
[0452] R130K;
[0453] I306A, G236S and F265Y;
[0454] I306A, R130K and G236S;
[0455] R130K and G236S;
[0456] I306A and R130K;
[0457] I306A, G236S and M237W;
[0458] I306A and F265Y;
[0459] I306A, R130K and G236S;
[0460] M237W;
[0461] I306A, R130K and F265Y;
[0462] R130K, G236S and M237W;
[0463] G236S and F265Y;
[0464] I306A and G236S;
[0465] R130K, G236S and F265Y;
[0466] I306A, R130K, G236S and F265Y;
[0467] I306A, G236S, M237W and F265Y;
[0468] I306A;
[0469] F265Y;
[0470] I306A and M237W;
[0471] R130K and M237W;
[0472] R130K and F265Y;
[0473] I306A, R130K and M237W;
[0474] I306A, M237W and F265Y;
[0475] R130K, M237W and F265Y;
[0476] G236S, M237W and F265Y;
[0477] M237W and F265Y;
[0478] I306A, R130K, M237W and F265Y;
[0479] R130K, G236S, M237W and F265Y;
[0480] I306A, R130K, G236S, M237W and F265Y;
[0481] I306A, R130K, G236S and D259A,
[0482] I306A, R130K, G236S and N258A;
[0483] I306A, R130K, G236S and N258D;
[0484] I306A, R130K, G236S and Q193A;
[0485] I306A, R130K, G236S and Q193E;
[0486] I306A, R130K, G236S, M237W and D259A;
[0487] I306A, R130K, G236S, M237W and N258A;
[0488] I306A, R130K, G236S, M237W and N258D;
[0489] I306A, R130K, G236S, M237W and Q193A; and
[0490] I306A, R130K, G236S, M237W and Q193E; preferably selected from
[0491] I306A, R130K, G236S and M237W
[0492] G236S and M237W
[0493] R130K increase(s) the selectivity for the production of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
Nucleic Acid Molecules
[0494] As discussed above, the present invention provides a nucleic acid molecule, coding for a protein or polypeptide according to the present invention.
[0495] As discussed above, the present invention provides nucleic acid molecules, coding for the proteins or polypeptides according to the present invention.
[0496] As discussed above, the present invention provides a nucleic acid molecule, coding for a domain or subunit of a polypeptide/protein according to the present invention.
[0497] Preferably the nucleic acid molecules of the invention comprise or consist of
the nucleic acid sequences of SEQ ID NO: 4 coding for the MPT domain (with polypeptide sequence SEQ ID NO: 1), SEQ ID NO: 5 coding for the AT domain (with polypeptide sequence SEQ ID NO: 2) and/or SEQ ID NO: 6 coding for the KS domain (with polypeptide sequence SEQ ID NO: 3), which comprise the respective nucleotide exchanges which lead to the amino acid substitution(s) of the present invention.
[0498] SEQ ID NO. 4 shows the nucleic acid sequence (part of GenBank: M30162.1 and J03936.1, split indicated by *) coding for the MPT domain (SEQ ID NO: 1):
TABLE-US-00004 TCTATCTTAGACATTGTCATTAACAACCCAGTTAACTTAACAATTCACTTCGGTGGTGAAAAGGGTAA GAGGATCAGAGAAAACTATTCTGCTATGATCTTTGAGACTATCGTGGATGGAAAATTGAAGACTGAAA AAATTTTCAAGGAAATTAATGAGCACAGTACTTCTTACACATTTAGATCTGAAAAAGGTTTATTGTCT GCTACTCAATTTACACAACCAGCTTTAACTTTGATGGAAAAAGCTGCTTTCGAAGACTTGAAATCTAA AGGTTTGATCCCAGCCGATGCTACTTTTGCTGGTCACTCTTTAGGTGAGTATGCTGCTTTGGCCTCTT TGGCTGATGTTATGTCTATCGAATCTTTAGTTGAAGTTGTGTTCTACAGAGGTATGACTATGCAAGTT GCTGTTCCAAGAGATGAGTTGGGCAGATCCAACTATGGTATGATTGCCATTAACCCAGGTAGAGTCGC TGCATCATTCTCTCAAGAAGCTTTGCAATATGTTGTTGAGAGAGTTGGTAAGAGAACCGGCTGGTTGG TTGAAATCGTCAACTACAACGTTGAAAACCAACAATATGTTGCAGCTGGTGATCTAAGAGCTTTAGAC ACCGTTACCAATGTTCTAAACTTCATCAAATTACAAAAAATTGATATTATTGAACTACAAAAGTCCTT ATCTTTGGAAGAAGTTGAAGGTCATTTGTTTGAGATCATTGACGAAGCTTCCAAGAAATCTGCTGTCA AGCCTCGCCCACTTAAATTGGAGAGAGGTTTTGCTTGTATCCCATTAGTTGGTATTTCTGTTCCTTTC CATTCCACCTACTTGATGAATGGTGTTAAACCATTCAAGAGTTTCTTGAAGAAGAATATCATAAAAGA AAATGTGAAGGTTGCTAGATTGGCCGGAAAGTACATTCCAAACTTGACTGCAAAACCATTCCAGGTTA CTAAGGAATATTTCCAGGACGTTTATGATTTGACTGGCTCCGAACCTATCAAGGAAATCATCGACAAC TGGGAAAAGTATGAACAATCC * ATGAAGCCGGAAGTTGAGCAAGAATTAGCTCATATTTTGCTAACTGAATTGTTAGCTTATCAATTTGC CTCTCCTGTGAGATGGATTGAAACTCAAGATGTTTTTITGAAGGATTTTAACACTGAAAGGGTTGTTG AAATCGGTCCTTCTCCAACTTTGGCTGGGATGGCTCAAAGAACCTTGAAGAATAAATACGAATCTTAC GATGCTGCTCTGTCTTTACATAGAGAAATCTTATGCTATTCG
[0499] SEQ ID NO: 5 shows the nucleic acid sequence (part of GenBank: M30162.1) coding for the AT domain (SEQ ID NO: 2)
TABLE-US-00005 ATGGACGCTTACTCCACAAGACCATTAACCCTATCTCACGGTTCTTTAG AGCACGTGCTTCTGGTACCAACCGCTTCATTTTTCATTGCTTCGCAATT ACAAGAACAATTTAATAAAATTTTGCCCGAACCCACTGAAGGGTTTGCT GCAGATGACGAGCCTACCACACCTGCTGAACTAGTGGGGAAATTCCTTG GCTACGTATCTTCTCTAGTCGAACCTTCCAAGGTCGGTCAATTCGATCA GGTCTTGAACCTTTGCTTAACAGAATTTGAAAACTGTTATTTAGAAGGC AATGACATTCACGCCTTGGCTGCTAAACTATTACAGGAAAACGACACAA CTTTAGTGAAGACTAAAGAACTAATTAAAAATTATATTACCGCCAGAAT AATGGCTAAGAGACCATTTGACAAAAAATCCAACTCTGCTCTTTTTAGG GCCGTCGGCGAGGGTAACGCACAATTGGTAGCCATTTTCGGTGGTCAAG GTAACACCGACGACTACTTTGAAGAATTGCGTGATCTATATCAAACTTA TCATGTCTTAGTGGGAGATTTAATCAAGTTCTCCGCTGAAACTTTAAGT GAACTGATTAGAACTACTTTAGATGCTGAAAAAGTCTTTACTCAAGGTT TAAACATATTGGAATGGTTGGAGAACCCTTCAAATACCCCAGACAAGGA CTATTTACTTTCCATTCCAATTTCATGCCCCTTAATTGGTGTCATTCAA TTGGCTCACTACGTAGTTACTGCCAAGCTTTTGGGTTTCACTCCAGGTG AGTTAAGATCTTACTTAAAAGGTGCTACAGGTCACTCTCAAGGTTTGGT TACTGCTGTCGCCATAGCTGAGACGGATTCCTGGGAATCCTTCTTCGTC TCCGTAAGAAAAGCAATTACTGTATTATTCTTCATCGGTGTTCGTTGTT ACGAAGCATACCCAAACACTTCCCTACCACCATCCATCTTGGAAGATTC CTTGGAAAACAATGAAGGTGTTCCATCTCCAATGTTGTCCATTTCCAAT CTAACTCAAGAACAAGTTCAAGACTATGTAAATAAGACTAACTCTCATT TGCCAGCTGGTAAACAAGTTGAAATTTCTCTAGTCAATGGTGCGAAGAA TCTAGTCGTATCGGGCCCACCACAATCATTATATGGTTTAAACTTGACT TTAAGAAAGGCCAAGGCCCCATCTGGACTGGATCAATCAAGAATCCCAT TCAGCGAAAGAAAATTGAAGTTCTCCAATAGGTTCTTACCTGTTGCATC ACCATTCCATTCCCATCTATTGGTTCCAGCTTCAGATTTGATTAACAAA GACTTAGTCAAAAACAATGTCAGCTTTAACGCTAAAGATATTCAAATCC CCGTTTACGACACTTTTGATGGTTCAGATCTAAGAGTCCTTTCAGGTTC CATTTCCGAGAGAATCGTCGACTGCATCATTAGATTACCTGTCAAATGG GAAACTACTACACAATTCAAAGCCACCCACATATTAGACTTTGGTCCAG GTGGAGCTTCCGGTTTAGGTGTTTTAACCCATCGTAATAAAGATGGTAC TGGTGTTCGTGTTATCGTTGCCGGTACTCTCGACATTAACCCAGATGAT GATTACGGATTCAAGCAAGAAATCTTTGATGTTACT
[0500] SEQ ID NO: 6 shows the nucleic acid sequence (part of GenBank: J03936.1) coding for the KS domain (SEQ ID NO: 3)
TABLE-US-00006 TTGGAAAGAGTTATTGTAGTTACCGGTTTTGCTGAAGTCGGCCCATGGG GTTCGGCCAGAACAAGATGGGAAATGGAAGCTTTTGGTGAATTTTCGTT GGAAGGTTGCGTTGAAATGGCCTGGATTATGGGCTTCATTTCATACCAT AACGGTAATTTGAAGGGTCGTCCATACACTGGTTGGGTTGATTCCAAAA CAAAAGAACCAGTTGATGACAAGGACGTTAAGGCCAAGTATGAAACATC AATCCTAGAACACAGTGGTATCAGATTGATCGAACCAGAGTTATTCAAT GGTTACAACCCAGAAAAGAAGGAAATGATTCAAGAAGTCATTGTCGAAG AAGACTTGGAACCATTTGAGGCTTCGAAGGAAACTGCCGAACAATTTAA ACACCAACATGGTGACAAAGTGGATATCTTCGAAATCCCAGAAACAGGA GAGTACTCTGTTAAGTTACTAAAGGGTGCCACTTTATACATTCCAAAGG CTTTGAGATTTGACCGTTTGGTTGCAGGTCAAATTCCAACTGGTTGGAA TGCTAAGACTTATGGTATCTCTGATGATATCATTTCTCAGGTTGACCCA ATCACATTATTCGTTTTGGTCTCTGTTGTGGAAGCATTTATTGCATCTG GTATCACCGACCCATACGAAATGTACAAATACGTACATGTTTCTGAGGT TGGTAACTGTTCTGGTTCTGGTATGGGTGGTGTTTCTGCCTTACGTGGT ATGTTTAAGGACCGTTTCAAGGATGAGCCTGTCCAAAATGATATTTTAC AAGAATCATTTATCAACACCATGTCCGCTTGGGTTAATATGTTGTTGAT TTCCTCATCTGGTCCAATCAAGACACCTGTTGGTGCCTGTGCCACATCC GTGGAATCTGTTGACATTGGTGTAGAAACCATCTTGTCTGGTAAGGCTA GAATCTGTATTGTCGGTGGTTACGATGATTTCCAAGAAGAAGGCTCCTT TGAGTTCGGTAACATGAAGGCCACTTCCAACACTTTGGAAGAATTTGAA CATGGTCGTACCCCAGCGGAAATGTCCAGACCTGCCACCACTACCCGTA ACGGTTTTATGGAAGCTCAAGGTGCTGGTATTCAAATCATCATGCAAGC TGATTTAGCTTTGAAGATGGGTGTGCCAATTTACGGTATTGTTGCCATG GCTGCTACCGCCACCCATAAGATTGGTAGATCTGTGCCAGCTCCAGGTA AGGGTATTTTAACCACTGCTCGTGAACACCACTCCAGTGTTAAGTATGC TTCACCAAACTTGAACATGAAGTACAGAAAGCGCCAATTGGTTACTCGT GAAGCTCAGATTAAAGATTGGGTAGAAAACGAATTGGAAGCTTTGAAGT TGGAGGCCGAAGAAATTCCAAGCGAAGACCAAAACGAGTTCTTACTTGA ACGTACCAGAGAAATCCACAACGAAGCTGAAAGTCAATTGAGAGCTGCA CAACAACAATGGGGTAACGACTTCTACAAGAGGGACCCACGTATTGCTC CATTGAGAGGAGCACTGGCTACTTACGGTTTAACTATTGATGACTTGGG TGTCGCTTCATTCCACGGTACATCCACAAAGGCTAATGACAAGAACGAA TCTGCCACAATTAATGAAATGATGAAGCATTTGGGTAGATCTGAAGGTA ATCCCGTCATTGGTGTTTTCCAAAAGTTCTTGACTGGTCATCCAAAGGG TGCTGCTGGTGCATGGATGATGAATGGTGCTTTGCAAATTCTAAACAGT GGTATTATTCCAGGTAACCGTAACGCTGATAACGTGGATAAGATCTTGG AGCAATTTGAATACGTCTTGTACCCATCCAAGACTTTAAAGACCGACGG TGTCAGAGCCGTGTCCATCACTTCTTTCGGTTTTGGTCAAAAGGGTGGT CAAGCTATTGTGGTTCATCCAGACTACTTATACGGTGCT
[0501] In one embodiment, the nucleic acid molecule of the present invention further comprises:
[0502] vector nucleic acid sequences, preferably expression vector sequences, and/or
[0503] promoter nucleic acid sequences and terminator nucleic acid sequences, and/or
[0504] comprises other regulatory nucleic acid sequence.
[0505] In one embodiment, the nucleic acid molecule of the present invention comprises dsDNA, ssDNA, cDNA, LNA, PNA, CNA, RNA or mRNA or combinations thereof.
[0506] The nucleic acid molecules according to the invention preferably comprise nucleic acid sequences, which are (except for the addition of the amino acid substitution(s) according to the invention) identical with the naturally occurring nucleic acid sequence or are codon-optimized for the use in a host cell.
[0507] The nucleic acid molecule used according to the present invention is preferably a nucleic acid expression construct.
[0508] Nucleic acid expression constructs according to the invention are expression cassettes comprising a nucleic acid molecule according to the invention, or expression vectors comprising a nucleic acid molecule according to the invention or an expression cassette, for example.
[0509] A nucleic acid expression construct preferably comprises regulatory sequences, such as promoter and terminator sequences, which are operatively linked with the nucleic acid sequence coding for the polypeptide(s) of the invention.
[0510] The nucleic acid expression construct may further comprise 5' and/or 3' recognition sequences and/or selection markers.
Host Cells
[0511] As discussed above, the present invention provides host cells containing a nucleic acid molecule according to the present invention.
[0512] Preferably, the host cells of the present invention express said nucleic acid molecule.
[0513] Preferably, a host cell according to the present invention is a bacterial cell.
[0514] The bacterial cell is more preferably a member of a genus selected from the group Corynebacterium, Mycobacterium, Escherichia, Nocordia, Bacillus, Clostridium, Pseudomonas, Lactobacillus or Leuconostoc.
[0515] The bacterial cell is more preferably a member of a species selected from the group of Corynebacterium glutamicum, Escherichia coli, Bacillus subtilis; Clostridium ljungdahlii, Pseudomonas putida; Lactobacillus bifermentans or Leuconostoc mesenteroides.
[0516] Preferably, a host cell according to the present invention is a fungus cell and more preferably a yeast cell.
[0517] The yeast cell is preferably a member of a genus selected from the group of Saccharomyces species, Kluyveromyces sp., Hansenula sp., Arxula sp., Rhodosporidium sp., Pichia sp. or Yarrowia sp.
[0518] The yeast cell is more preferably a member of a species selected from the group of S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, K. fragilis, H. polymorpha, P. pastoris and Y. lipolytica,
such as S. cerevisiae, K. lactis, H. polymorpha, P. pastoris, K. marxianus, or Y. lipolytica.
[0519] In a preferred embodiment, the host cell belongs to the species Saccharomyces cerevisiae.
[0520] Preferably, a host cell according to the present invention is an algae cell.
[0521] The algae cell is more preferably a member of a genus selected from the group Chlamydomonas, Chlorella, Haematococcus, Dunaliella, Nannochloropsis, Thalassiosira, Phaeodactylum, Porphyridium or Scenedesmus
[0522] The algae cell is more preferably a member of a species selected from the group of Chlamydomonas reinhardtii or Haematococcus pluvialis.
[0523] Preferably, the host cell (preferably yeast cell) has an elevated overall production of short fatty acids, CoA esters of short fatty acids, ethyl esters of short fatty acids, esters of short fatty acids with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12) compared to a cell not containing a nucleic acid molecule according to the present invention.
[0524] In a preferred embodiment, the host cell has an increased yield or increased selectivity in the production of C.sub.6 fatty acids, C.sub.6 fatty acid CoA esters, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, and/or enzyme bound C.sub.6 fatty acids compared to a cell not containing a nucleic acid molecule according to the present invention.
[0525] The selectivity for the production of C.sub.6 fatty acids, C.sub.6 CoA esters and/or enzyme bound C.sub.6 fatty acids compared to wild type is increased where the share of C.sub.6 makes up at least 30%, or preferably at least 40% or 50% or 70% or 80% or 90% of the detected chain length between C.sub.6 and C.sub.12.
[0526] As discussed above, in particular proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0527] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0528] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0529] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0530] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0531] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0532] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0533] R130K (in the MPT domain on SEQ ID NO: 1);
[0534] I306A (in the AT domain on SEQ ID NO: 2), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0535] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0536] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: I);
[0537] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0538] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0539] G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0540] R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0541] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0542] I306A (in the AT domain on SEQ ID NO: 2), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0543] I306A (in the AT domain on SEQ ID NO: 2);
[0544] M237W (in the KS domain on SEQ ID NO: 3);
[0545] F265Y (in the KS domain on SEQ ID NO: 3);
[0546] I306A (in the AT domain on SEQ ID NO: 2) and M237W (in the KS domain on SEQ ID NO: 3);
[0547] R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0548] R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0549] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0550] I306A (in the AT domain on SEQ ID NO: 2), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0551] R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0552] G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0553] M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0554] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0555] R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0556] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0557] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258A (in the KS domain on SEQ ID NO: 3);
[0558] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258D (in the KS domain on SEQ ID NO: 3);
[0559] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258A (in the KS domain on SEQ ID NO: 3);
[0560] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258D (in the KS domain on SEQ ID NO: 3); preferably selected from
[0561] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0562] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0563] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0564] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3)
[0565] G236S and M237W (in the KS domain on SEQ ID NO: 3), more preferably
[0566] I306A and G236S,
[0567] I306A and F265Y,
[0568] I306A, G236S and M237W,
[0569] I306A, R130K and G236S, increase(s) the selectivity for the production of C.sub.6 fatty acids, C.sub.6 CoA, C.sub.6 fatty acid ethyl esters, C.sub.6 fatty acid esters with other metabolites, esters and/or enzyme bound C.sub.6 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0570] In a preferred embodiment, the host cell has an increased yield or increased selectivity in the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to a cell not containing a nucleic acid molecule according to the present invention.
[0571] The selectivity for the production of C.sub.8 fatty acids, C.sub.8 CoA esters and/or enzyme bound C.sub.8 fatty acids compared to wild type is increased where the share of C.sub.8 makes up at least 30%, or preferably at least 40% or 50% or 70% or 80% or 89% of the detected chain length between C.sub.6 and C.sub.12.
[0572] As discussed above, in particular proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0573] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3)
[0574] R130K (in the MPT domain on SEQ ID NO: 1);
[0575] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0576] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0577] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: 1);
[0578] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0579] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0580] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0581] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0582] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0583] M237W (in the KS domain on SEQ ID NO: 3);
[0584] I306A (in the AT domain on SEQ ID NO: 2), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0585] G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0586] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0587] R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3)
[0588] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0589] I306A (in the AT domain on SEQ ID NO: 2), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0590] I306A (in the AT domain on SEQ ID NO: 2);
[0591] F265Y (in the KS domain on SEQ ID NO: 3);
[0592] I306A (in the AT domain on SEQ ID NO: 2) and M237W (in the KS domain on SEQ ID NO: 3);
[0593] R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0594] R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0595] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0596] I306A (in the AT domain on SEQ ID NO: 2), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0597] R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0598] G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0599] M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0600] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0601] R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0602] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0603] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and D259A (in the KS domain on SEQ ID NO: 3);
[0604] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and D259A (in the KS domain on SEQ ID NO: 3);
[0605] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258A (in the KS domain on SEQ ID NO: 3);
[0606] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258A (in the KS domain on SEQ ID NO: 3);
[0607] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258D (in the KS domain on SEQ ID NO: 3);
[0608] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258D (in the KS domain on SEQ ID NO: 3);
[0609] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0610] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0611] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193E (in the KS domain on SEQ ID NO: 3);
[0612] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193E (in the KS domain on SEQ ID NO: 3); preferably selected from
[0613] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3)
[0614] R130K (in the MPT domain on SEQ ID NO: 1);
[0615] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0616] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0617] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: 1);
[0618] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0619] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0620] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0621] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3); more preferably,
[0622] I306A, R130K and F265Y;
[0623] R130K;
[0624] I306A, R130K, G236S and M237W;
[0625] R130K and G236S;
[0626] I306A and R130K;
[0627] G236S and M237W;
[0628] I306A, R130K and G236S; increase(s) the selectivity for the production of C.sub.8 fatty acids, C.sub.8 fatty acid CoA esters, C.sub.8 fatty acid ethyl esters, C.sub.8 fatty acid esters with other metabolites, and/or enzyme bound C.sub.8 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s).
[0629] In a preferred embodiment, the host cell has an increased yield or increased selectivity in the production of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to a cell not containing a nucleic acid molecule according to the present invention.
[0630] As discussed above, in particular proteins or polypeptides of the present invention, wherein the amino acid substitution(s) is/are selected from
[0631] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0632] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0633] R130K (in the MPT domain on SEQ ID NO: 1);
[0634] I306A (in the AT domain on SEQ ID NO: 2), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0635] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0636] R130K (in the MPT domain on SEQ ID NO: 1) and G236S (in the KS domain on SEQ ID NO: 3);
[0637] I306A (in the AT domain on SEQ ID NO: 2) and R130K (in the MPT domain on SEQ ID NO: 1);
[0638] I306A (in the AT domain on SEQ ID NO: 2), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0639] I306A (in the AT domain on SEQ ID NO: 2) and F265Y (in the KS domain on SEQ ID NO: 3);
[0640] M237W (in the KS domain on SEQ ID NO: 3);
[0641] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0642] R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0643] G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0644] I306A (in the AT domain on SEQ ID NO: 2) and G236S (in the KS domain on SEQ ID NO: 3);
[0645] R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0646] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and F265Y (in the KS domain on SEQ ID NO: 3);
[0647] I306A (in the AT domain on SEQ ID NO: 2), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0648] I306A (in the AT domain on SEQ ID NO: 2);
[0649] F265Y (in the KS domain on SEQ ID NO: 3);
[0650] I306A (in the AT domain on SEQ ID NO: 2) and M237W (in the KS domain on SEQ ID NO: 3);
[0651] R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0652] R130K (in the MPT domain on SEQ ID NO: 1) and F265Y (in the KS domain on SEQ ID NO: 3);
[0653] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1) and M237W (in the KS domain on SEQ ID NO: 3);
[0654] I306A (in the AT domain on SEQ ID NO: 2), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0655] R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0656] G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0657] M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0658] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0659] R130K (in the MPT domain on SEQ ID NO: I), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0660] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and F265Y (in the KS domain on SEQ ID NO: 3);
[0661] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and D259A (in the KS domain on SEQ ID NO: 3);
[0662] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and D259A (in the KS domain on SEQ ID NO: 3);
[0663] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258A (in the KS domain on SEQ ID NO: 3);
[0664] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258A (in the KS domain on SEQ ID NO: 3);
[0665] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and N258D (in the KS domain on SEQ ID NO: 3);
[0666] I306A (in the AT domain on SEQ ID NO: 2), RI 30K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and N258D (in the KS domain on SEQ ID NO: 3);
[0667] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0668] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193 .ANG. (in the KS domain on SEQ ID NO: 3);
[0669] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and Q193E (in the KS domain on SEQ ID NO: 3);
[0670] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S, M237W and Q193E (in the KS domain on SEQ ID NO: 3); preferably selected from
[0671] I306A (in the AT domain on SEQ ID NO: 2), R130K (in the MPT domain on SEQ ID NO: 1), G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0672] G236S and M237W (in the KS domain on SEQ ID NO: 3);
[0673] R130K (in the MPT domain on SEQ ID NO: 1); increase(s) the selectivity for the production of C.sub.10 to C.sub.12 fatty acids, C.sub.10 to C.sub.12 fatty acid CoA esters, C.sub.10 to C.sub.12 fatty acid ethyl esters, C.sub.10 to C.sub.12 fatty acid esters with other metabolites, and/or enzyme bound C.sub.10 to C.sub.12 fatty acids compared to wild type polypeptide(s) or the polypeptide without such amino acid substitution(s). Methods and Uses for Producing Fatty Acids and/or Biofuels and/or Flavoring Substances and/or Fine Chemicals
[0674] As discussed above, the present invention provides a method for the production of short fatty acids, CoA esters of short fatty acids, short fatty acid ethyl esters, short fatty acid esters with other metabolites, and/or enzyme bound short fatty acids (C.sub.6 to C.sub.12).
[0675] Said method comprises the expression of a nucleic acid molecule according to the present invention, preferably in a host cell according to the present invention.
[0676] As discussed above, the present invention provides a method for the production of
[0677] biofuels,
[0678] flavoring compounds or substances
[0679] and/or
[0680] fine chemicals.
[0681] Said method comprises the expression of a nucleic acid molecule according to the present invention, preferably in a host cell according to the present invention.
[0682] Biofuels which can be produced with the method are, for example, short alkanes, short alkenes, short alkynes, short esters or alcohols.
[0683] Flavoring compounds or substances which can be produced with the method are, for example, fatty acids esterified with short alcohols or esters from short fatty acids (C.sub.6 to C.sub.12).
[0684] Fine chemicals which can be produced with the method are, for example, natural compounds, where preferably short fatty acids (C.sub.6 to C.sub.12) or their derivatives (such as CoA esters, methyl/ethyl esters, esters with other metabolites, alcohols) are used as building block(s).
[0685] As discussed above, the present invention provides the use of
[0686] a protein or polypeptide according to the present invention,
[0687] a nucleic acid molecule according to the present invention, or
[0688] a host cell according to the present invention, for
[0689] the bulk production of short fatty acids (C.sub.6 to C.sub.12), CoA esters of short fatty acids, short fatty acid ethyl esters, short fatty acid esters with other metabolites, enzyme bound short fatty acids; or
[0690] the specific production of short fatty acids (C.sub.6 or C.sub.8), C.sub.6-CoA esters or C.sub.8-CoA esters, fatty acid (C.sub.6 or C.sub.8) ethyl esters, fatty acid (C.sub.6 or C.sub.8) esters with other metabolites, enzyme bound short fatty acids (C.sub.6 or C.sub.8).
[0691] In particular, the present invention provides the use of a protein or polypeptide according to the present invention, a nucleic acid molecule according to the present invention, or a host cell according to the present invention, for
[0692] the bulk production of short fatty acids (C.sub.6 to C.sub.12),
[0693] the specific production of C.sub.6 fatty acids,
[0694] the specific production of C.sub.8 fatty acids,
[0695] the bulk production of CoA esters of short fatty acids (C.sub.6 to C.sub.12),
[0696] the specific production of C.sub.6 fatty acid CoA esters,
[0697] the specific production of C.sub.8 fatty acid CoA esters,
[0698] the bulk production of ethyl esters of short fatty acids (C.sub.6 to C.sub.12),
[0699] the specific production of C.sub.6 fatty acid ethyl esters,
[0700] the specific production of C.sub.8 fatty acid ethyl esters,
[0701] the bulk production of short fatty acids (C.sub.6 to C.sub.u) esters with other metabolites,
[0702] the specific production of C.sub.6 fatty acid esters with other metabolites,
[0703] the specific production of C.sub.8 fatty acid esters with other metabolites,
[0704] the bulk production of enzyme bound short fatty acids (C.sub.6 to C.sub.12),
[0705] the specific production of enzyme bound C.sub.6 fatty acids, or
[0706] the specific production of enzyme bound C.sub.8 fatty acids. As discussed above, the present invention further provides the use of
[0707] a protein or polypeptide according to the present invention,
[0708] a nucleic acid molecule according to the present invention, or
[0709] a host cell according to the present invention, for
[0710] the production of biofuels, such as short alkanes, short alkenes, short alkynes, short esters or alcohols,
[0711] the production of fine chemicals, such as natural compounds where preferably short fatty acids (C.sub.6 to C.sub.12) or their derivatives (such as CoA esters, methyl/ethyl esters, esters with other metabolites, alcohols) are used as building block(s), or
[0712] the production of flavoring compounds or substances, such as esters from short fatty acids (C.sub.6 to C.sub.12).
Further Description of Preferred Embodiments
[0713] The study shown here, is the first one reported where by rational design S. cerevisiae strains were created to produce short fatty acids. It was not only possible to show significant increase in their overall production with the highest yield of 118 mg/L (27-fold increase over the wild type) in regular YPD but also the production of specific chain lengths was demonstrated: One strain optimized for hexanoic acid (C.sub.6) showed yields of 20 mg/L corresponding to 90% of the detected short FA (C.sub.6 to C.sub.12) in that strain. For specific octanoic acid (C.sub.8) production, a share of 89% was shown at an output level of 48 mg/L.
[0714] Abstract
[0715] In the present invention, the production of short fatty acids was achieved by a structural based rational design, where only key residues in FAS were changed to impose a system of chain length control.
[0716] Besides elevated overall levels of short fatty acids (118 mg/L, a 27-fold increase over the wild type) also the specific production of C.sub.6 (20 mg/L corresponding to 90% of the detected short FA, C.sub.6 to C.sub.12) and C.sub.8 (48 mg/L, 89% of detected short FA) was possible.
[0717] Results
[0718] For the production of short FA in S. cerevisiae, a .DELTA.fas1 .DELTA.fas2 strain was created. Two heterozygotic strains with one deletion each, were mated and then sporulated to gain the double knockout strain. The two chains of FAS were transformed into cells on two separate low copy vectors (pRS315 and pRS313 respectively) under control of their natural promoters and terminators (Chirala 1992). The plasmid FAS system was then the only source of de-novo fatty acids.
[0719] The FAS itself, only produces FA as CoA esters and not as free acids. For S. cerevisiae several proteins were reported recently (see e.g. Knight et al., 2014) which putatively hydrolyze the short CoA esters to the free FA. These putative TEs can also contribute to the distinct product distribution due to their own substrate specificity. For the actual quantification, the media was analyzed because short chain free FA produced by S. cerevisiae are exported from the cell (Leber & da Silva, 2014).
[0720] For the production of short fatty acids, the fatty acids cycle was manipulated in all enzymatic centers that are potentially involved in chain length control (see FIG. 1), the condensation domain (KS) and the transferases (MPT and AT). While the modifying domains (KR, DH and ER) can have different affinities towards intermediates of different lengths, they do not play the key role in determining the product length.
1. Ketoacyl Synthase (KS) Domain:
[0721] The KS domain catalyzes the condensation step in the fatty acid synthesis, meaning the actual elongation of intermediates. Mechanistically, the catalyzed reaction works in a ping-pong mechanism: In a first step, the acyl-intermediate is loaded from the ACP into the KS where it binds covalently to C291 (ping step). When the ACP returns to the KS with a bound malonyl, the latter is decarboxylated. The produced carboxylanion reacts with the acyl chain in the KS and thereby cleaves it off from the active center of the KS (pong step).
[0722] The mutations introduced into the KS domain (see FIG. 4) for chain length control are influencing the ping step of the reaction by restricting the loading of acyl chains beyond a certain length. As an elongation is less likely (or inhibited), more short FA are produced.
[0723] In the first position, a G236S mutation was introduced. This mutation has been linked to enhanced resistance to Cerulenin, a common FAS inhibitor. In S. cerevisiae strains with this mutation used for Sake production, an increased level of C.sub.6 and its ester derivatives was reported (Aritomi et al., 2004; see also JP 2002/027989 A).
[0724] In comparison with the wild type, the G236S strain shows a significant increase of C.sub.6 with 15.3 mg/L on average, a 9-fold increase over the wild type (see FIG. 2).
[0725] As a second position in the KS, the neighboring M237 was mutated. Comparison of the structural data of the FAS with a bound Cerulenin (Johansson et al., 2008) and the FAS without the latter (Leibundgut et al., 2007), indicated two conformations for M237: a) It was found in a position pointing directly into the KS binding channel and b) pushed aside by Cerulenin which is mimicking a bound acyl substrate. A gatekeeper function of a methionine in this position in the KS binding channel has also previously postulated to explain the bimodal product spectrum found for the HsmtKAS (Christensen et al., 2007). Most likely the reported lower binding for C.sub.8-ACP (Zhang et al., 2005) can be linked to the rearrangement of the methionine residue as well.
[0726] Our intention was to enhance the gatekeeper effect of this crucial position and hinder the elongation of acyl intermediates beyond certain length by replacing it with a bigger, bulkier residue. A M237W mutation was chosen.
[0727] The double mutant G236S-M237W was tested and increased levels of C.sub.6 (with 19.9 mg/L, 12-fold increase over wild type) and C.sub.8 (32.7 mg/L, 56-fold increase over wild type) were found (FIG. 2).
[0728] Also, a third promising position in the KS was mutated based on sequence alignments with organisms known to produce C.sub.6 at least as an intermediate, such as Aspergillus parasiticus and Aspergillus flavus (see e.g. Hitchman et al., 2001). The F235Y mutation is located on the opposite side of the G236S-M237W mutation in the binding channel of the KS (see FIG. 4) but is believed to also impede binding of acyl chains beyond C.sub.6. Effects of the measured G236S-F237Y double mutant strain could hardly be interpreted since its growth was highly inhibited resulting in very low short FA output. Its results in combination with mutations in the transferases are reported below.
[0729] The positions Q193, N258 and D259 are also located at the surface of the KS domain, where they interfere with interaction of the ACP and KS domain. The mutated positions are to lower probability of this interaction. Consequently, an elongation of produced fatty acids is less likely, because intermediates (which are bound to the ACP domain) are not elongated directly and the chances of an early export by the promiscuous MPT domain are increased, leading to shorter fatty acids (FA).
2. Malonyl-/Palmitoyl-Transferase (MPT) Domain:
[0730] In the yeast FAS systems, the MPT domain is responsible for malonyl-CoA loading and also for unloading of products (typically C.sub.16 and C.sub.18) as CoA esters. Similar to the KS domain, transferases in FAS also work in ping-pong mechanism. A substrate is first loaded from a CoA into the transferase where it binds covalently and is then transferred onto the ACP domain or vice versa. In the MPT specifically, both compound groups, malonyl substrates and acyl products, are transferred via this mechanism and essentially compete in the ping step for one residue, S104, in the active center. The MPT's responsibility in product cleave off made it a target for engineering.
[0731] Inherently, the MPT domain is a transferase with a generally broad substrate spectrum, which includes short acyl chains as well. The approach of destabilizing malonyl binding in the MPT and thus, shifting the equilibrium of substrate loading and product unloading in favor of the latter was implemented. It was previously shown in structural studies for other, distant related transferases, that a central arginine stabilizes the carboxyl group of malonyl (see e.g. Bunkoczi et al., 2009). In the present study, a R130K mutation was introduced in the MPT domain, which was believed to have two effects on chain length control: 1) Products could be released more easily since the active site is less often occupied with malonyl. 2) Malonyl as an elongation substrate is loaded less likely into the enzyme, which would have a comparable effect to an overall decrease in malonyl concentration; a condition which has been linked to the production of shorter fatty acids (see e.g. Kawaguchi et al., 1980).
[0732] The effects of the R130K mutation in MPT domain on the product spectrum are dramatic (FIG. 2): The overall production of short FA made up from mostly C.sub.8 was 100 mg/L, a 23-fold increase over the wild type.
3. Acetyl Transferase (AT) Domain:
[0733] In the S. cerevisiae FAS, the loading of acetyl onto the enzyme is catalyzed by a second transferase, the acetyl transferase (AT). Despite the differences in substrate specificity, all acyl transferases show high homologies. Since the mentioned studies (see e.g. Bunkoczi et al., 2009) also lead to an increased acetyl binding, similar engineering on the AT domain was considered, potentially leading to an increased acetyl/malonyl ratio on the enzyme, which is beneficial for the production of short FA.
[0734] In the present study, an I306A mutation was introduced. As a second desirable effect, an acquired ability to transfer short acyl chains could open up a new way for product release.
4. Combinations of Mutations:
[0735] The mutations in different domains were also combined in several variations (FIG. 2), not only leading to increased overall amounts, but also several yeast strains with high chain length specificity. See Table 1.
[0736] Table 1 also gives an indication which chain length is favored with the introduction of a particular mutation. For some mutations, it can be clearly seen, that if an increased short FA output was found, this was mainly due to a higher yield in C.sub.6 as found when the G236S or the I306A mutation is added to an existing combination. The introduction of the M237W or the R130K mutation, however, rather leads to an increase in C.sub.8, if an increase in short FA was detected at all.
[0737] A few combinations and their effects are listed here:
[0738] The highest total yield was achieved with the combination of I306A-R130K-G236S (AT MPT KS mutant), producing a total amount of 118 mg/L.
[0739] For the specific production of C.sub.6, the double mutant I306A-G236S (AT KS mutant) showed best results with 20 mg/L of C.sub.6 accounting for 90% of the measured short FA (C.sub.6 to C.sub.12).
[0740] A similar specificity, but for the production for C.sub.8, was possible with the triple mutant I306A-R130K-F265Y (AT MPT KS mutant) with a yield of 48 mg/L for C.sub.8 specifically representing 89% of its short FA output.
[0741] With the I306A-R130K-G236S-N258A construct but a slightly different vector (promoter exchanged ADH2 promotor and buffered medium, 100 mM K2HPO4/KH2PO4), the total yield was also very high with 344 mg/L.
[0742] Discussion
[0743] In our study, we focused on one aspect of reaction control of fatty acid production, the chain length regulation, and its rational manipulation in the enzyme responsible for its production, the fatty acid synthase. Only key residues were to be modified providing a minimally invasive system for the in vivo production of short fatty acids in S. cerevisiae. The FAS was brought into a fas1 fas2 knockout strain via a vector, but under its natural promoter and only on a low copy plasmid, mimicking natural conditions. Hence, the system was not optimized for best overall FA production but instead was rather to ensure highest possible control, also providing the ability to directly map the relations between mutations and yield in short FA.
[0744] In order to alter the product spectrum, the FAS was modified in all active centers potentially regulating chain length:
1) In the KS domain located on FAS 2, six residues were altered in order to limit elongation of intermediate products (G236S, M237W, F265Y, Q193A/E, N258A/D, D259A). 2) In the MPT domain located on FAS 1, loading of malonyl used for elongation was artificially limited and product release accordingly facilitated (R130K). 3) In the AT domain located on FAS 1, binding channels were broadened to increase acetyl influx or more likely, providing a new way for short chain product release (I306A).
[0745] The engineering presented here can be understood as metabolic engineering. Fluxes were changed rather than only the ability of one individually domain to process a certain reaction. On the FAS itself, twelve reaction steps can be catalyzed (when transferase steps are included) and this is not considering that eight of them are iteratively repeated several times until a certain chain length is reached. In this complex network of reactions which are essentially influencing each other, we were able to alter affinities and consequently change the kinetics and reaction rates with the introduction of concise mutations.
[0746] An expansive influence on the FA chain length could be shown. The effects were measured with the result of different overall amounts of short FA and a highest overall yield of 118 mg/L. The increase is 27-fold higher than in the wild type, that clearly only produces short FA as a minor byproduct in the range of 4.3 mg/L. Also distinct and controllable selectivity of chain lengths was demonstrated. For specific production of C.sub.6, the I306A-G236S mutant was favorable (20 mg/L, 90% share C.sub.6), for C.sub.8, the I306A-R130K-F265Y mutant respectively (48 mg/L, 89% share of C.sub.8).
[0747] The immediate products of the FAS, the acyl CoA esters, were not quantified but instead the extracellular free FA. This implies proteins with thioesterase activity hydrolyzing acyl CoA esters intracellularly (Knight et al., 2014). These proteins and fatty acid transporters potentially contribute to distinct product spectra.
[0748] The scope of the study was to modulate chain length in a non-invasive approach. This means that the fas1 fas2 knockout strain was complemented with fas1 and fas2 variants set under their native promoters and terminators.
[0749] In our experiments, the viability of the cells was influenced by the specific mutations in the FAS. The data suggest that some of the mutations significantly influenced the fatty acid spectrum, i.e. decreasing intracellular C.sub.16/C.sub.18 fatty acid (CoA esters) levels, so that growth was affected. Such an impact on the viability of cells by some of our mutations is supported by supplementation studies with C.sub.18:1, that generally restored normal growth behavior (Table 2). This implies that the system is highly complex and short chain fatty acid production has to be adapted to host cell architecture.
Table 1: Increase/Decrease of Yield after Introduction of Mutations.
[0750] To quantify how beneficial a mutation is, the strain without the mutation (first line) is compared to the same strain with the mutation (second line). By dividing the second value by the first value, the increase a mutation adds to the yield (for C.sub.6, C.sub.8 and total short FA) is given as a factor ("x-fold increase"). Accordingly, values above 1 (in bold) are equivalent to a positive, higher yield; for values below 1, the yield dropped with the introduction of the mutation.
TABLE-US-00007 TABLE 1 Increase/Decrease of yield after introduction of mutations. C.sub.6 C.sub.8 total (C.sub.6 to C.sub.12) Mutation (mg/L) (mg/L) (mg/L) G236S wild type 1.7 0.6 4.3 (KS domain) G236S 15.3 3.9 21.6 X-fold increase 9.1 6.7 5.0 R130K 15.7 80.2 100.1 R130K G236S 9.4 26.7 39.5 X-fold increase 0.6 0.3 0.4 M237W G236S 15.3 3.9 21.6 (KS domain) G236S M237W 19.9 32.7 57.2 X-fold increase 1.3 8.3 2.6 I306A G236S 20.4 1.0 22.8 G236S M237W I306A 37.1 25.6 65.9 X-fold increase 1.8 25.2 2.9 I306A R130K G236S 52.1 63.1 118.2 G236S M237W R130K 13.0 77.1 104.1 I306A X-fold increase 0.2 1.2 0.9 F265Y I306A R130K 10.1 25.1 37.4 (KS domain) I306A R130K F265Y 2.2 47.9 53.9 X-fold increase 0.2 1.9 1.4 I306A G236S 15.3 3.9 21.6 (AT domain) G236S I306A 20.4 1.0 22.8 X-fold increase 1.3 0.3 1.1 G236S M237W 19.9 32.7 57.2 G236S M237W I306A 37.1 25.6 65.9 X-fold increase 1.9 0.8 1.2 R130K 15.7 80.2 100.1 R130K I306A 10.1 25.1 37.4 X-fold increase 0.6 0.3 0.4 R130K wild type 1.7 0.6 4.3 (MPT R130K 15.7 80.2 100.1 domain) X-fold increase 9.4 137.3 23.4 G236S 15.3 3.9 21.6 G236S R130K 9.4 26.7 39.5 X-fold increase 0.6 6.8 1.8 I306A G236S 20.4 1.0 22.8 I306A G236S R130K 52.1 63.1 118.2 X-fold increase 2.6 62.2 5.2 G236S M237W I306A 37.1 25.6 65.9 G236S M237W I306A 13.0 77.1 104.1 R130K X-fold increase 0.4 3.0 1.6
[0751] The following examples and drawings illustrate the present invention without, however, limiting the same thereto.
BRIEF DESCRIPTION OF THE DRAWINGS
[0752] FIG. 1. Fatty acid cycle and its manipulation.
[0753] The FAS carries modifications in the KS, AT and MPT domain to produce shorter fatty acids (instead of its native product, typically C.sub.16- or C.sub.18-CoA) from acetyl-CoA, malonyl-CoA and NADPH. The KS mutations (G236S, M237W and F265Y) were constructed to restrict the loading of substrates beyond a certain length (indicated with a dotted line leading to the KS domain) and thus leading to the formation of shorter products. The AT (with the I306A mutation) was to enhance the loading of acetyl-CoA (indicated with the bold arrow at the AT domain) and/or act as a transferase to cleave off short chain products (dashed arrow from acyl products through the AT domain), a reaction not found in wild type FAS. The MPT mutation R130K was introduced to shift the balance in binding of malonyl and acyl chains in favor of the latter. Both the lowered malonyl loading (indicated by an arrow with a smaller tip at the MPT domain) as well as the easier acyl chain release add to an increase of short fatty acids. S. cerevisiae FAS naturally produces CoA esters, which are hydrolyzed by thioesterases, if they are shorter than a certain length. The free FA are then transported out of the cell into the media, from which they are extracted for their analysis.
[0754] FIG. 2. Product spectra of selected mutated strains in YPD.
[0755] For the measurements of the product spectra, cultures of S. cerevisiae were grown for 48 h at 30.degree. C., the media extracted and later quantified via GC-FID. Error bars shown here reflect the standard deviation from two independent experiments (beginning from separate transformations into S. cerevisiae). The strain carrying the I306A-R130K-G236S-M11251W mutations and the strain with the I306A-R130K-F265Y mutations (both marked #) only grew to approximately one third of the regular cell density of the rest.
[0756] FIG. 3. Growing curve of manipulated Yeast strains.
[0757] For selected strains, the cell density was monitored at several time points.
[0758] FIG. 4. Binding channel of the KS domain with mutation sites.
[0759] Here, the KS domain from the S. cerevisiae FAS (light grey) is shown in cartoon depiction with important residues shown in stick representation (based on PDB code 2VKZ). The active center C291 is located on the left with a bound Cerulenin molecule (dark grey), a known FAS inhibitor mimicking a bound acyl. The binding channel extends to the right, where three mutation sites, G236S, M1250W and F265Y are shown with their initial amino acids.
[0760] FIG. 5. Product Spectra at additional time points of selected strains.
[0761] Besides the regular product spectra after 48 h, additional measurements were performed for selected strains after 12 h and 24 h.
[0762] FIGS. 6A-6E. Glucose and Ethanol concentration over time.
[0763] For selected strains, the medium was monitored at several time points during the 48 h cultivation. The amount of remaining glucose and produced ethanol in the fermentation medium was measured with HPLC.
[0764] FIGS. 7A-7E. Acetate and Glycerol concentration over time.
[0765] For the same selected strains as in FIGS. 6A-6E, also the acetate and glycerol concentrations in the medium were analyzed by HPLC.
[0766] FIG. 8. Product spectrum of a further mutated strain in YPD.
[0767] The product spectrum of a strain carrying the I306A-R130K-G236S-D259A mutations is shown in comparison to the wild type. In this case, the medium was buffered to pH 6.5 (100 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4) and the promotor was exchanged for both the wild type and the construct to ADH2. For the measurements of the product spectrum, cultures of S. cerevisiae were grown at 30.degree. C., cell growth was hindered and after 72 h cell density was only 5.0 (OD.sub.600), the media extracted and later quantified via GC-FID. Error bars shown here reflect the standard deviation from three independent results (beginning from separate clones of S. cerevisiae).
[0768] FIG. 9. Product spectrum of a further mutated strain in YPD.
[0769] The product spectrum of a strain carrying the I306A-R130K-G236S-N258A mutations is shown in comparison to the wild type. In this case, the medium was buffered to pH 6.5 (100 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4) and the promotor was exchanged for both the wild type and the construct to ADH2).
[0770] For the measurements of the product spectrum, cultures of S. cerevisiae were grown at 30.degree. C., cell growth was hindered and after 72 h cell density was only 5.0 (OD.sub.600), the media extracted and later quantified via GC-FID. Error bars shown here reflect the standard deviation from three independent results (beginning from separate clones of S. cerevisiae).
EXAMPLES
Example 1
1. Materials and Methods
1.1 Description Yeast Strain
[0771] The haploid S. cerevisiae strain BY.PK1238_1A_KO, used in this work, has a BY background and the reading frames of FAS1 and FAS2 are each replaced by a kanMX4 cassette, resulting in a clean knock out of FAS I and antibiotic resistance against Geneticin. The exact genotype is Mata; ura3.DELTA.0; his3.DELTA.0; leu2.DELTA.0; TRP1; lys2.DELTA.0; MET15; fas1::uptag-kanMX4-downtag; fas2::uptag-kanMX4-downtag.
1.2 Vector Description
[0772] The vectors used in this work are centromeric pRS shuttle vectors of types pRS313 and pRS315 (Sikorski & Meter, 1989) with single copy number and HIS3 and LEU2 auxotrophy marker, respectively. FAS1 or mutations thereof were always provided on pRS315, while FAS2 or mutations thereof were always provided on pRS313, each regulated by its according native promoter (995 bp upstream for FAS1 and 480 bp upstream for FAS2) (Chirala 1992). Terminator sequences were set to 295 bp and 258 bp, respectively, downstream of the open reading frames. Cloning was always done in E. coli using the Infusion HD cloning kit (Clontech, Mountain View, USA).
[0773] Wild type FAS1 and FAS2 genes were assembled from several fragments, which were amplified from S. cerevisiae genomic DNA, into pRS vectors using BamHI and Sail restriction sites. Exact chromosomal coordinates including promoter and terminator sequences according to strain S288C are for FAS1 (YKL182w): Chr XI 99676-107121 and for FAS2 (YPL231w): ChrXVI 108172-114573.
1.3 Primers
[0774] For the introduction of mutations by site-directed mutagenesis, the primers are listed below. The mutation site is indicated in bold typing, while the overlap between the primers is underlined.
[0775] For FAS 1 variants:
TABLE-US-00008 I306A forward SEQ ID NO: 7 5'-TTCTTCGCTGGTGTTCGTTGTTACGAAGCATACCCAAACACTTCC- 3' I306A reverse SEQ ID NO: 8 5'-ACACCAGCG AAG AATAATACAGTAATTGCTTTTCTTACGGAGA CG-3' R13 OK forward SEQ ID NO: 9 5'-AGTTGTGTTCTACAAAGGTATGACTATGCAAGTTGCTGTTCC-3' R130K reverse SEQ ID NO: 10 5'-CATAGTCATACCTTTGTAGAACACAACTTCAACTAAAGATTCGATAG AC-3'
[0776] For FAS 2 variants:
TABLE-US-00009 G236S forward SEQ ID NO: 11 5'-TCTGGTTCTTCTATGGGTGGTGTTTCTGCCTTACG-3' G236S reverse SEQ ID NO: 12 5'-CATAGAAGAACCAGAACAGTTACCAACCTCAGAAACATGTACG-3' M237W forward SEQ ID NO: 13 5'-TCTGGTTCTTCTTGGGGTGGTGTTTCTGCCTTACG-3' M237W reverse SEQ ID NO: 14 5'-CCAAGAAGAACCAGAACAGTTACCAACCTCAGAAACATGTACG-3' F265Y forward SEQ ID NO: 15 5'-ATTTTACAAGAATCA TAT ATCAACACCATGTCCGC-3' F265Y reverse SEQ ID NO: 16 5'-TGATTCTTGTAAAATATCATTTTGGACAGGC-3'
1.4 Transformation
[0777] For yeast transformation, approximately 1 .mu.g of each plasmid DNA was co-transformed following a modified lithium acetate protocol (Schiestl & Gietz, 1989). A 5 mL overnight culture of strain BY.PK1238_1A_KO in YPD (1% yeast extract, 2% peptone, both produced by BD, Difco Laboratories, Sparks, USA; 2% dextrose, purchased from Roth, Karlsruhe, Germany) containing 200 .mu.g/mL Geneticin disulfate, free fatty acids (myristic, palmitic and stearic acid, each 50 .mu.g/mL) and 1% Tween20 grown at 30.degree. C. and 200 rpm was used to inoculate a main culture in the same medium. After shaking at 30.degree. C. and 200 rpm to OD.sub.600=0.8, a volume of 5 mL of this culture was harvested by centrifugation (3000 rcf, 5 min, 24.degree. C.). The cells were washed by resuspending in 1 mL water and centrifuged again. After resuspension in lithium acetate solution (0.1 M), cells were incubated for 5 min at 24.degree. C. and centrifuged (5000 rcf, 15 s, 24.degree. C.), before the transformation mix was added (240 .mu.L PEG 1,500 solution (50%), 76 .mu.L water, 36 .mu.L lithium acetate solution (1.0 M), 5.0 .mu.L single stranded DNA solution from salmon testis (10 mg/mL), 2 .mu.L of each plasmid DNA solution). The cell suspension was mixed well and incubated for 30 min at 30.degree. C. followed by 20 min at 42.degree. C. After pelleting the cells by centrifugation (4000 rcf, 15 s, 4.degree. C.), they were washed with 1 mL water, pelleted again (4000 rcf, 15 s, 4.degree. C.) and resuspended in 100 .mu.L water. For selection of the yeast transformants, the cell suspension was spread on SCD -ura; -leu agar plates containing 200 .mu.g/mL Geneticin disulfate, free fatty acids (myristic, palmitic and stearic acid, each 50 .mu.g/mL) and 1% Tween20.
1.5 Cultures for Product Analysis
[0778] For the product analysis, several colonies of the S. cerevisiae strains were picked and united in one pre-culture (5 ml YPD with 200 .mu.g/mL Geneticin disulfate, 50 mg/ml final concentration). After shaking at 200 rpm at 30.degree. C. overnight, the OD.sub.600 was measured. The main culture (50 ml YPD with 200 .mu.g/mL Geneticin disulfate, 50 mg/ml final concentration) was inoculated to OD.sub.600=0.1 and shaken for 48 h at 200 rpm and 30.degree. C. Before further processing, the OD.sub.600 was measured again.
[0779] For samples with long FA supplementation, C.sub.151 and Tergitol NP-40 (solution in water, 70%) were added to all cultures to a final concentration of 1 mM or 1% in the case of Tergitol.
1.6 Sample Processing
[0780] For FA extraction, a protocol similar to a previously published one (Leber & da Silva, 2014) was used: First cells were spun down at 3,500 ref for 15 min. The supernatant was aliquoted in 10 ml portions and 0.2 mg of the internal standard, heptanoic acid (C.sub.7), was added. After acidification with 1 ml HCl (1 M), 2.5 ml of a mixture of equal amounts of methanol and chloroform were added. The samples were shaken vigorously for 5 min and then centrifuged again at 3 500 rcf for 10 min. The chloroform layer was transferred to a new vial and any residual water removed. The liquid was then fully evaporated in a SpeedVac.
[0781] For methylation a previously published protocol was used (Ichihara & Fukubayashi, 2010).
1.7 Determination of Free Fatty Acid by Gas Chromatography (GC)
[0782] The resulting fatty acid methyl esters dissolved in hexane (Ichihara & Fukubayashi, 2010), were measured with a Perkin Elmer Clarus 400 gaschromatograph (Perkin Elmer, Rodgau, Germany) equipped with an Elite FFAP capillary column (30 m.times.0.25 mm, film thickness: 0.25 urn; Perkin Elmer, Rodgau, Germany) and a flame ionization detector (Perkin Elmer, Rodgau, Germany). 1 .mu.L of the sample was analyzed after split injection (10 mL/min) and helium as carrier gas. The temperatures of the injector and detector were 200.degree. C. and 250.degree. C., respectively. The following temperature program was applied: 50.degree. C. for 5 min, increase of 10.degree. C./min to 120.degree. C. (hold for 5 min), increase of 15.degree. C./min to 180.degree. C. (hold for 10 min), and increase of 20.degree. C./min to 220.degree. C. for 37 min.
1.8 Metabolite Analysis by HPLC
[0783] For quantification of glucose, ethanol, glycerol and acetate 450 .mu.L cell-free samples were mixed with 50 .mu.L of 50% (w/v) 5-sulfosalicylic acid, vigorously shaken and centrifuged (4.degree. C., 5 min, 13 000 rcf). The supernatant was analyzed with an UHPLC+ system (Dionex UltiMate 3000, Thermo Scientific, Dreieich, Germany) equipped with a HyperREZ XP Carbohydrate H+ 8 .mu.m column. To detect the substrates a refractive index detector (Thermo Shodex RI-101) was used. Separation was carried out at 65.degree. C. with 5 mM sulfuric acid as mobile phase (flow rate of 0.6 ml/min). Five standards (mixtures of D-glucose, ethanol, glycerol and acetate with concentrations of 0.05-2% (w/v)) were analyzed for quantification of the different compounds.
1.9 Determination of Cell Density
[0784] The cell density in a liquid culture was measured with an Ultrospec 2100 pro spectrophotometer (GE Healthcare, USA) by determination of the optical density at 600 nm (OD.sub.600).
2. Results
[0785] For the production of short FA in S. cerevisiae, a .DELTA.fas1 .DELTA.fas2 strain was created. Two heterozygotic strains with one deletion each, were mated and then sporulated to gain the double knockout strain. The two chains of FAS were transformed into cells on two separate low copy vectors (pRS315 and pRS313 respectively) under control of their natural promoters and terminators (Chirala 1992). The plasmid FAS system was then the only source of de-novo fatty acids.
2.1 Short FA Yield in YPD Supplemented with C.sub.18:1
[0786] In some strains, growth was severely inhibited in regular YPD media, most likely because the plasmid FAS system did not produce enough long fatty acids necessary for cell growth. As an alternative, strains were also tested in YPD media supplemented with oleic acid (C.sub.18:1, 1 mM) where all strains showed similar growth (Table 2). The reestablishment in growth is proof that an insufficient production of long chains prevented cells from growing before.
2.2 Vitality Parameters
[0787] In our in vivo study, the cells themselves were closely monitored. The cell density was measured for all samples at the end of the growing period and the wet cell pellet weight was noted (Table 2). In addition, for selected samples OD.sub.600 was recorded at several time points (FIG. 3). For the growth in regular YPD, three groups can be distinguished: Strains that showed regular growth, ones with reduced growth and a group, were only an extremely low or no detectable growth could be seen. When the media was supplemented with C.sub.18:1 (1 mM) all strains showed similar growth.
[0788] In order to test the theory, that reduced growth for the I306A-R130K-G236S-M237W mutant could derive from a strong initial production of C.sub.8 eventually inhibiting further growth, product spectra of selected strains were measured after 12 h and 24 h in addition to the regular measurements after 48 h (FIG. 5). While cell density of the I306A-R130K-G236S-M237W mutant reached its stationary phase already after 24 h instead of a later time (as for the R130K mutant for example, FIG. 3), the FA production follows a similar pattern for both the samples.
TABLE-US-00010 TABLE 2 Cell density (OD.sub.600 and wet pellet weight) after 48 h. Samples in regular Samples in YPD with YPD C.sub.18:1 wet cell wet cell OD.sub.600 pellet (g) OD.sub.600 pellet (g) wild type 24.7 1.1 17.4 1.3 G236S 21.9 1.1 19.2 1.4 G236S M237W 15.6 1.0 19.5 1.5 I306A 23.1 1.1 19.2 1.4 I306A G236S M237W 15.9 1.1 18.8 1.4 R130K G236S M237W 3.5 0.2 22.0 1.5 I306A R130K G236S M237W 5.4 0.5 20.4 1.7 R130K 20.7 1.1 24.1 1.4 I306 R130K 22.5 1.2 20.3 1.5 I306A G236S 15.6 1.3 18.9 1.4 R130K G236S 10.2 0.9 19.6 1.4 I306A R130K G236S 14.0 1.1 18.3 1.3 G236S F265Y 0.1 0.3 17.5 1.4 I306A G236S F265Y 0.3 0.3 16.7 1.3 R130K G236S F265Y 0.1 0.3 15.8 1.4 I306A R130K G236S F265Y 0.1 0.3 17.0 1.5 I306A F265Y 15.6 1.2 16.9 1.3 I306A G236S M237W F265Y -- * -- * 18.2 1.4 I306A F265W -- * -- * 17.7 1.3 I306A G236S F265W -- * -- * 21.8 1.4 I306A G236S M237W F265W -- * -- * 18.4 1.3 I306A R130K F265Y 4.3 0.6 21.1 1.4 I306A R130K G236S M237W -- * -- * 20.5 1.3 F265Y I306A R130K F265W -- * -- * 23.6 1.4 I306A R130K G236S F265W -- * -- * 15.8 1.4 I306A R130K G236S M237W -- * -- * 21.1 1.3 F265W
[0789] Just before further processing, the OD.sub.600 was measured for selected samples, both when they were grown in regular YPD and in YPD supplemented with C.sub.18:1 (1 mM). For the growth in regular YPD, samples could be divided into three groups: regular growth (white background), reduced growth (light gray background) and very little/no growth (dark gray background). In YPD supplemented with C.sub.18:1 all samples showed nearly the same densities. Also, the wet pellet weight was noted. It is, however, prone to errors since residual media that is stuck to the tube can make the results less reliable. For samples marked with an asterisk (*), no main culture was grown, after the preculture already showed no significant growth.
2.3 Glucose Consumption, Ethanol Production, FA Production on Ethanol
[0790] For cells grown with YPD media the glucose consumption and ethanol synthesis was measured. For all tested strains (FIGS. 6A-6E) glucose is entirely consumed after 20 h, whereas the synthesis of ethanol starts after approx. 10 h, when a low glucose concentration (<1.2% (wt/vol)) is measurable. Corresponding to this, the amount of glycerol and acetate (FIGS. 7A-7E) increases after 10 h of fermentation, which can be compared to the strain with the wild type FAS.
Example 2
1. Materials and Methods
[0791] For this example, materials and methods were the same as in Example 1, if not stated otherwise.
1.1 Vector Description
[0792] See Example 1. For constructs carrying any of the mutations Q193A, Q193E, N258A, N258D, D259A, the ADH2 promotor was used. For comparison, also one wild type construct was cloned with this promotor and used as a reference when constructs with this particular promotor were tested.
1.2 Primers
[0793] For the introduction of the point mutations in a PCR, the primers are listed below. The PCR products were then cloned into the vector containing the ADH2 promotor via homologous recombination. fw=forward, rv=reverse
TABLE-US-00011 FAS2-D259A_rv SEQ ID NO: 17 TCTTGTAAAATAGCATTTTGGACAGGCTCATCCTTGAAACGGTCCTTAA AC FAS2-D259A-fw SEQ ID NO: 18 CCTGTCCAAAATGCTATTTTACAAGAATCATTTATCAACACCATGTCCG CTTGGG FAS2-N258A_rv SEQ ID NO: 19 TCTTGTAAAATATCAGCTTGGACAGGCTCATCCTTGAAACGGTCCTTAA AC FAS2-N258A_fw SEQ ID NO: 20 CCTGTCCAAGCTGATATTTTACAAGAATCATTTATCAACACCATGTCCG CTTGGG FAS2_Q193A_rv SEQ ID NO: 21 GAATAATGTGATTGGGTCAACCGCAGAAATGATATCATCAGAGATACCA TAAGTC FAS2_Q193A_fw SEQ ID NO: 22 CTGCGGTTGACCCAATCACATTATTCGTTTTGGTCTCTGTTGTGGAAG FAS2_Q193E_rv SEQ ID NO: 23 GAATAATGTGATTGGGTCAACCTCAGAAATGATATCATCAGAGATACCA TAAGTC FAS2_Q193E_fw SEQ ID NO: 24 CTGAGGTTGACCCAATCACATTATTCGTTTTGGTCTCTGTTGTGGAAG FAS2_N258D_rv SEQ ID NO: 25 TCTTGTAAAATATCATCTTGGACAGGCTCATCCTTGAAACGGTCCTTAA AC FAS2_N258D_fw SEQ ID NO: 26 CCTGTCCAAGATGATATTTTACAAGAATCATTTATCAACACCATGTCCG CTTGGG
[0794] The following primers were designed for homologous recombination of the cut vector and the ADH2 promotor.
TABLE-US-00012 pRS315-pADH2 SEQ ID NO: 27 CAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGAGCTGGCAAAA CGTAGGGGCAAACAAACG pADH2-Fas2 SEQ ID NO: 28 GCAAAATATGAGCTAATTCTTGCTCAACTTCCGGCTTCATTGTGTATTA CGATATAGTTAATAG Fas2-pADH2 SEQ ID NO: 29 GCATACAATCAACTATCAACTATTAACTATATCGTAATACACAATGAAG CCGGAAGTTGAGCAAGAATTAG pRS42_hxt7 SEQ ID NO: 30 CACACAGGAAACAGCTATGAC
1.3 Cultures for Product Analysis
[0795] In contrast to the procedure described in Example 1, the main culture was buffered to pH 6.5 ((100 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4). Cell cultivation was 72 hours.
[0796] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.
REFERENCES
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[0798] Beld J, Lee D J, & Burkart M D (2015) Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering. Molecular BioSystems 11(1):38-59.
[0799] Bunkoczi G, et al. (2009) Structural Basis for Different Specificities of Acyltransferases Associated with the Human Cytosolic and Mitochondrial Fatty Acid Synthases. Chemistry & Biology 16(6):667-675.
[0800] Chirala S S (1992) Coordinated regulation and inositol-mediated and fatty acid-mediated repression of fatty acid synthase genes in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America 89(21):10232-10236.
[0801] Choi Y J & Lee S Y (2013) Microbial production of short-chain alkanes. Nature 502(7472):571-574.
[0802] Christensen C E, Kragelund B B, von Wettstein-Knowles P, & Henriksen A (2007) Structure of the human .beta.-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase. Protein Science 16(2):261-272.
[0803] Grininger M (2014) Perspectives on the evolution, assembly and conformational dynamics of fatty acid synthase type I (FAS I) systems. Current Opinion in Structural Biology 25(0):49-56.
[0804] Hitchman T S, et al. (2001) Hexanoate Synthase, a Specialized Type I Fatty Acid Synthase in Aflatoxin B1 Biosynthesis. Bioorganic Chemistry 29(5):293-307.
[0805] Ichihara Ki & Fukubayashi Y (2010) Preparation of fatty acid methyl esters for gas-liquid chromatography. Journal of Lipid Research 51(3):635-640.
[0806] Jenni S, et al. (2007) Structure of Fungal Fatty Acid Synthase and Implications for Iterative Substrate Shuttling. Science 316(5822):254-261.
[0807] Johansson P, et al. (2008) Inhibition of the fungal fatty acid synthase type I multienzyme complex. Proceedings of the National Academy of Sciences 105(35):12803-12808.
[0808] Kawaguchi A, Arai K, Seyama Y, Yamakawa T, & Okuda S (1980) Substrate Control of Termination of Fatty Acid Biosynthesis by Fatty Acid Synthetase from Brevibacterium ammoniagenes TI. The Journal of Biochemistry 88(2):303-306.
[0809] Knight M J, Bull I D, & Curnow P (2014) The yeast enzyme Eht1 is an octanoyl-CoA:ethanol acyltransferase that also functions as a thioesterase. Yeast 31(12):463-474.
[0810] Leber C & Da Silva N A (2014) Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids. Biotechnology and Bioengineering 111(2):347-358.
[0811] Leber C, Polson B, Fernandez-Moya R, & Da Silva N A (2015) Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae. Metabolic Engineering 28(0):54-62.
[0812] Leibundgut M, Jenni S, Frick C, & Ban N (2007) Structural Basis for Substrate Delivery by Acyl Carrier Protein in the Yeast Fatty Acid Synthase. Science 316(5822):288-290.
[0813] Liu H, et al. (2012) Production of extracellular fatty acid using engineered Escherichia coli. Microbial Cell Factories 11.
[0814] Peralta-Yahya, P. P., Zhang, F., del Cardayre, S. B. & Keasling, J. D. (2012) Microbial engineering for the production of advanced biofuels. Nature 488: 320-328.
[0815] Runguphan W & Keasling J D (2014) Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals. Metabolic Engineering 21(0):103-113.
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[0818] Tehlivets O, Scheuringer K, & Kohlwein S D (2007) Fatty acid synthesis and elongation in yeast. Regulation of Lipid Metabolism in Yeast 1771(3):255-270.
[0819] Zhang L, Joshi A K, Hofmann J, Schweizer E, & Smith S (2005) Cloning, Expression, and Characterization of the Human Mitochondrial .beta.-Ketoacyl Synthase: COMPLEMENTATION OF THE YEAST CEM1 KNOCK-OUT STRAIN. Journal of Biological Chemistry 280(13):12422-12429.
Sequence CWU
1
1
301429PRTSaccharomyces cerevisiae 1Ser Ile Leu Asp Ile Val Ile Asn Asn Pro
Val Asn Leu Thr Ile His1 5 10
15Phe Gly Gly Glu Lys Gly Lys Arg Ile Arg Glu Asn Tyr Ser Ala Met
20 25 30Ile Phe Glu Thr Ile Val
Asp Gly Lys Leu Lys Thr Glu Lys Ile Phe 35 40
45Lys Glu Ile Asn Glu His Ser Thr Ser Tyr Thr Phe Arg Ser
Glu Lys 50 55 60Gly Leu Leu Ser Ala
Thr Gln Phe Thr Gln Pro Ala Leu Thr Leu Met65 70
75 80Glu Lys Ala Ala Phe Glu Asp Leu Lys Ser
Lys Gly Leu Ile Pro Ala 85 90
95Asp Ala Thr Phe Ala Gly His Ser Leu Gly Glu Tyr Ala Ala Leu Ala
100 105 110Ser Leu Ala Asp Val
Met Ser Ile Glu Ser Leu Val Glu Val Val Phe 115
120 125Tyr Arg Gly Met Thr Met Gln Val Ala Val Pro Arg
Asp Glu Leu Gly 130 135 140Arg Ser Asn
Tyr Gly Met Ile Ala Ile Asn Pro Gly Arg Val Ala Ala145
150 155 160Ser Phe Ser Gln Glu Ala Leu
Gln Tyr Val Val Glu Arg Val Gly Lys 165
170 175Arg Thr Gly Trp Leu Val Glu Ile Val Asn Tyr Asn
Val Glu Asn Gln 180 185 190Gln
Tyr Val Ala Ala Gly Asp Leu Arg Ala Leu Asp Thr Val Thr Asn 195
200 205Val Leu Asn Phe Ile Lys Leu Gln Lys
Ile Asp Ile Ile Glu Leu Gln 210 215
220Lys Ser Leu Ser Leu Glu Glu Val Glu Gly His Leu Phe Glu Ile Ile225
230 235 240Asp Glu Ala Ser
Lys Lys Ser Ala Val Lys Pro Arg Pro Leu Lys Leu 245
250 255Glu Arg Gly Phe Ala Cys Ile Pro Leu Val
Gly Ile Ser Val Pro Phe 260 265
270His Ser Thr Tyr Leu Met Asn Gly Val Lys Pro Phe Lys Ser Phe Leu
275 280 285Lys Lys Asn Ile Ile Lys Glu
Asn Val Lys Val Ala Arg Leu Ala Gly 290 295
300Lys Tyr Ile Pro Asn Leu Thr Ala Lys Pro Phe Gln Val Thr Lys
Glu305 310 315 320Tyr Phe
Gln Asp Val Tyr Asp Leu Thr Gly Ser Glu Pro Ile Lys Glu
325 330 335Ile Ile Asp Asn Trp Glu Lys
Tyr Glu Gln Ser Met Lys Pro Glu Val 340 345
350Glu Gln Glu Leu Ala His Ile Leu Leu Thr Glu Leu Leu Ala
Tyr Gln 355 360 365Phe Ala Ser Pro
Val Arg Trp Ile Glu Thr Gln Asp Val Phe Leu Lys 370
375 380Asp Phe Asn Thr Glu Arg Val Val Glu Ile Gly Pro
Ser Pro Thr Leu385 390 395
400Ala Gly Met Ala Gln Arg Thr Leu Lys Asn Lys Tyr Glu Ser Tyr Asp
405 410 415Ala Ala Leu Ser Leu
His Arg Glu Ile Leu Cys Tyr Ser 420
4252551PRTSaccharomyces cerevisiae 2Met Asp Ala Tyr Ser Thr Arg Pro Leu
Thr Leu Ser His Gly Ser Leu1 5 10
15Glu His Val Leu Leu Val Pro Thr Ala Ser Phe Phe Ile Ala Ser
Gln 20 25 30Leu Gln Glu Gln
Phe Asn Lys Ile Leu Pro Glu Pro Thr Glu Gly Phe 35
40 45Ala Ala Asp Asp Glu Pro Thr Thr Pro Ala Glu Leu
Val Gly Lys Phe 50 55 60Leu Gly Tyr
Val Ser Ser Leu Val Glu Pro Ser Lys Val Gly Gln Phe65 70
75 80Asp Gln Val Leu Asn Leu Cys Leu
Thr Glu Phe Glu Asn Cys Tyr Leu 85 90
95Glu Gly Asn Asp Ile His Ala Leu Ala Ala Lys Leu Leu Gln
Glu Asn 100 105 110Asp Thr Thr
Leu Val Lys Thr Lys Glu Leu Ile Lys Asn Tyr Ile Thr 115
120 125Ala Arg Ile Met Ala Lys Arg Pro Phe Asp Lys
Lys Ser Asn Ser Ala 130 135 140Leu Phe
Arg Ala Val Gly Glu Gly Asn Ala Gln Leu Val Ala Ile Phe145
150 155 160Gly Gly Gln Gly Asn Thr Asp
Asp Tyr Phe Glu Glu Leu Arg Asp Leu 165
170 175Tyr Gln Thr Tyr His Val Leu Val Gly Asp Leu Ile
Lys Phe Ser Ala 180 185 190Glu
Thr Leu Ser Glu Leu Ile Arg Thr Thr Leu Asp Ala Glu Lys Val 195
200 205Phe Thr Gln Gly Leu Asn Ile Leu Glu
Trp Leu Glu Asn Pro Ser Asn 210 215
220Thr Pro Asp Lys Asp Tyr Leu Leu Ser Ile Pro Ile Ser Cys Pro Leu225
230 235 240Ile Gly Val Ile
Gln Leu Ala His Tyr Val Val Thr Ala Lys Leu Leu 245
250 255Gly Phe Thr Pro Gly Glu Leu Arg Ser Tyr
Leu Lys Gly Ala Thr Gly 260 265
270His Ser Gln Gly Leu Val Thr Ala Val Ala Ile Ala Glu Thr Asp Ser
275 280 285Trp Glu Ser Phe Phe Val Ser
Val Arg Lys Ala Ile Thr Val Leu Phe 290 295
300Phe Ile Gly Val Arg Cys Tyr Glu Ala Tyr Pro Asn Thr Ser Leu
Pro305 310 315 320Pro Ser
Ile Leu Glu Asp Ser Leu Glu Asn Asn Glu Gly Val Pro Ser
325 330 335Pro Met Leu Ser Ile Ser Asn
Leu Thr Gln Glu Gln Val Gln Asp Tyr 340 345
350Val Asn Lys Thr Asn Ser His Leu Pro Ala Gly Lys Gln Val
Glu Ile 355 360 365Ser Leu Val Asn
Gly Ala Lys Asn Leu Val Val Ser Gly Pro Pro Gln 370
375 380Ser Leu Tyr Gly Leu Asn Leu Thr Leu Arg Lys Ala
Lys Ala Pro Ser385 390 395
400Gly Leu Asp Gln Ser Arg Ile Pro Phe Ser Glu Arg Lys Leu Lys Phe
405 410 415Ser Asn Arg Phe Leu
Pro Val Ala Ser Pro Phe His Ser His Leu Leu 420
425 430Val Pro Ala Ser Asp Leu Ile Asn Lys Asp Leu Val
Lys Asn Asn Val 435 440 445Ser Phe
Asn Ala Lys Asp Ile Gln Ile Pro Val Tyr Asp Thr Phe Asp 450
455 460Gly Ser Asp Leu Arg Val Leu Ser Gly Ser Ile
Ser Glu Arg Ile Val465 470 475
480Asp Cys Ile Ile Arg Leu Pro Val Lys Trp Glu Thr Thr Thr Gln Phe
485 490 495Lys Ala Thr His
Ile Leu Asp Phe Gly Pro Gly Gly Ala Ser Gly Leu 500
505 510Gly Val Leu Thr His Arg Asn Lys Asp Gly Thr
Gly Val Arg Val Ile 515 520 525Val
Ala Gly Thr Leu Asp Ile Asn Pro Asp Asp Asp Tyr Gly Phe Lys 530
535 540Gln Glu Ile Phe Asp Val Thr545
5503650PRTSaccharomyces cerevisiae 3Leu Glu Arg Val Ile Val Val Thr
Gly Phe Ala Glu Val Gly Pro Trp1 5 10
15Gly Ser Ala Arg Thr Arg Trp Glu Met Glu Ala Phe Gly Glu
Phe Ser 20 25 30Leu Glu Gly
Cys Val Glu Met Ala Trp Ile Met Gly Phe Ile Ser Tyr 35
40 45His Asn Gly Asn Leu Lys Gly Arg Pro Tyr Thr
Gly Trp Val Asp Ser 50 55 60Lys Thr
Lys Glu Pro Val Asp Asp Lys Asp Val Lys Ala Lys Tyr Glu65
70 75 80Thr Ser Ile Leu Glu His Ser
Gly Ile Arg Leu Ile Glu Pro Glu Leu 85 90
95Phe Asn Gly Tyr Asn Pro Glu Lys Lys Glu Met Ile Gln
Glu Val Ile 100 105 110Val Glu
Glu Asp Leu Glu Pro Phe Glu Ala Ser Lys Glu Thr Ala Glu 115
120 125Gln Phe Lys His Gln His Gly Asp Lys Val
Asp Ile Phe Glu Ile Pro 130 135 140Glu
Thr Gly Glu Tyr Ser Val Lys Leu Leu Lys Gly Ala Thr Leu Tyr145
150 155 160Ile Pro Lys Ala Leu Arg
Phe Asp Arg Leu Val Ala Gly Gln Ile Pro 165
170 175Thr Gly Trp Asn Ala Lys Thr Tyr Gly Ile Ser Asp
Asp Ile Ile Ser 180 185 190Gln
Val Asp Pro Ile Thr Leu Phe Val Leu Val Ser Val Val Glu Ala 195
200 205Phe Ile Ala Ser Gly Ile Thr Asp Pro
Tyr Glu Met Tyr Lys Tyr Val 210 215
220His Val Ser Glu Val Gly Asn Cys Ser Gly Ser Gly Met Gly Gly Val225
230 235 240Ser Ala Leu Arg
Gly Met Phe Lys Asp Arg Phe Lys Asp Glu Pro Val 245
250 255Gln Asn Asp Ile Leu Gln Glu Ser Phe Ile
Asn Thr Met Ser Ala Trp 260 265
270Val Asn Met Leu Leu Ile Ser Ser Ser Gly Pro Ile Lys Thr Pro Val
275 280 285Gly Ala Cys Ala Thr Ser Val
Glu Ser Val Asp Ile Gly Val Glu Thr 290 295
300Ile Leu Ser Gly Lys Ala Arg Ile Cys Ile Val Gly Gly Tyr Asp
Asp305 310 315 320Phe Gln
Glu Glu Gly Ser Phe Glu Phe Gly Asn Met Lys Ala Thr Ser
325 330 335Asn Thr Leu Glu Glu Phe Glu
His Gly Arg Thr Pro Ala Glu Met Ser 340 345
350Arg Pro Ala Thr Thr Thr Arg Asn Gly Phe Met Glu Ala Gln
Gly Ala 355 360 365Gly Ile Gln Ile
Ile Met Gln Ala Asp Leu Ala Leu Lys Met Gly Val 370
375 380Pro Ile Tyr Gly Ile Val Ala Met Ala Ala Thr Ala
Thr Asp Lys Ile385 390 395
400Gly Arg Ser Val Pro Ala Pro Gly Lys Gly Ile Leu Thr Thr Ala Arg
405 410 415Glu His His Ser Ser
Val Lys Tyr Ala Ser Pro Asn Leu Asn Met Lys 420
425 430Tyr Arg Lys Arg Gln Leu Val Thr Arg Glu Ala Gln
Ile Lys Asp Trp 435 440 445Val Glu
Asn Glu Leu Glu Ala Leu Lys Leu Glu Ala Glu Glu Ile Pro 450
455 460Ser Glu Asp Gln Asn Glu Phe Leu Leu Glu Arg
Thr Arg Glu Ile His465 470 475
480Asn Glu Ala Glu Ser Gln Leu Arg Ala Ala Gln Gln Gln Trp Gly Asn
485 490 495Asp Phe Tyr Lys
Arg Asp Pro Arg Ile Ala Pro Leu Arg Gly Ala Leu 500
505 510Ala Thr Tyr Gly Leu Thr Ile Asp Asp Leu Gly
Val Ala Ser Phe His 515 520 525Gly
Thr Ser Thr Lys Ala Asn Asp Lys Asn Glu Ser Ala Thr Ile Asn 530
535 540Glu Met Met Lys His Leu Gly Arg Ser Glu
Gly Asn Pro Val Ile Gly545 550 555
560Val Phe Gln Lys Phe Leu Thr Gly His Pro Lys Gly Ala Ala Gly
Ala 565 570 575Trp Met Met
Asn Gly Ala Leu Gln Ile Leu Asn Ser Gly Ile Ile Pro 580
585 590Gly Asn Arg Asn Ala Asp Asn Val Asp Lys
Ile Leu Glu Gln Phe Glu 595 600
605Tyr Val Leu Tyr Pro Ser Lys Thr Leu Lys Thr Asp Gly Val Arg Ala 610
615 620Val Ser Ile Thr Ser Phe Gly Phe
Gly Gln Lys Gly Gly Gln Ala Ile625 630
635 640Val Val His Pro Asp Tyr Leu Tyr Gly Ala
645 65041287DNASaccharomyces cerevisiae 4tctatcttag
acattgtcat taacaaccca gttaacttaa caattcactt cggtggtgaa 60aagggtaaga
ggatcagaga aaactattct gctatgatct ttgagactat cgtggatgga 120aaattgaaga
ctgaaaaaat tttcaaggaa attaatgagc acagtacttc ttacacattt 180agatctgaaa
aaggtttatt gtctgctact caatttacac aaccagcttt aactttgatg 240gaaaaagctg
ctttcgaaga cttgaaatct aaaggtttga tcccagccga tgctactttt 300gctggtcact
ctttaggtga gtatgctgct ttggcctctt tggctgatgt tatgtctatc 360gaatctttag
ttgaagttgt gttctacaga ggtatgacta tgcaagttgc tgttccaaga 420gatgagttgg
gcagatccaa ctatggtatg attgccatta acccaggtag agtcgctgca 480tcattctctc
aagaagcttt gcaatatgtt gttgagagag ttggtaagag aaccggctgg 540ttggttgaaa
tcgtcaacta caacgttgaa aaccaacaat atgttgcagc tggtgatcta 600agagctttag
acaccgttac caatgttcta aacttcatca aattacaaaa aattgatatt 660attgaactac
aaaagtcctt atctttggaa gaagttgaag gtcatttgtt tgagatcatt 720gacgaagctt
ccaagaaatc tgctgtcaag cctcgcccac ttaaattgga gagaggtttt 780gcttgtatcc
cattagttgg tatttctgtt cctttccatt ccacctactt gatgaatggt 840gttaaaccat
tcaagagttt cttgaagaag aatatcataa aagaaaatgt gaaggttgct 900agattggccg
gaaagtacat tccaaacttg actgcaaaac cattccaggt tactaaggaa 960tatttccagg
acgtttatga tttgactggc tccgaaccta tcaaggaaat catcgacaac 1020tgggaaaagt
atgaacaatc catgaagccg gaagttgagc aagaattagc tcatattttg 1080ctaactgaat
tgttagctta tcaatttgcc tctcctgtga gatggattga aactcaagat 1140gtttttttga
aggattttaa cactgaaagg gttgttgaaa tcggtccttc tccaactttg 1200gctgggatgg
ctcaaagaac cttgaagaat aaatacgaat cttacgatgc tgctctgtct 1260ttacatagag
aaatcttatg ctattcg
128751653PRTSaccharomyces cerevisiae 5Ala Thr Gly Gly Ala Cys Gly Cys Thr
Thr Ala Cys Thr Cys Cys Ala1 5 10
15Cys Ala Ala Gly Ala Cys Cys Ala Thr Thr Ala Ala Cys Cys Cys
Thr 20 25 30Ala Thr Cys Thr
Cys Ala Cys Gly Gly Thr Thr Cys Thr Thr Thr Ala 35
40 45Gly Ala Gly Cys Ala Cys Gly Thr Gly Cys Thr Thr
Cys Thr Gly Gly 50 55 60Thr Ala Cys
Cys Ala Ala Cys Cys Gly Cys Thr Thr Cys Ala Thr Thr65 70
75 80Thr Thr Thr Cys Ala Thr Thr Gly
Cys Thr Thr Cys Gly Cys Ala Ala 85 90
95Thr Thr Ala Cys Ala Ala Gly Ala Ala Cys Ala Ala Thr Thr
Thr Ala 100 105 110Ala Thr Ala
Ala Ala Ala Thr Thr Thr Thr Gly Cys Cys Cys Gly Ala 115
120 125Ala Cys Cys Cys Ala Cys Thr Gly Ala Ala Gly
Gly Gly Thr Thr Thr 130 135 140Gly Cys
Thr Gly Cys Ala Gly Ala Thr Gly Ala Cys Gly Ala Gly Cys145
150 155 160Cys Thr Ala Cys Cys Ala Cys
Ala Cys Cys Thr Gly Cys Thr Gly Ala 165
170 175Ala Cys Thr Ala Gly Thr Gly Gly Gly Gly Ala Ala
Ala Thr Thr Cys 180 185 190Cys
Thr Thr Gly Gly Cys Thr Ala Cys Gly Thr Ala Thr Cys Thr Thr 195
200 205Cys Thr Cys Thr Ala Gly Thr Cys Gly
Ala Ala Cys Cys Thr Thr Cys 210 215
220Cys Ala Ala Gly Gly Thr Cys Gly Gly Thr Cys Ala Ala Thr Thr Cys225
230 235 240Gly Ala Thr Cys
Ala Gly Gly Thr Cys Thr Thr Gly Ala Ala Cys Cys 245
250 255Thr Thr Thr Gly Cys Thr Thr Ala Ala Cys
Ala Gly Ala Ala Thr Thr 260 265
270Thr Gly Ala Ala Ala Ala Cys Thr Gly Thr Thr Ala Thr Thr Thr Ala
275 280 285Gly Ala Ala Gly Gly Cys Ala
Ala Thr Gly Ala Cys Ala Thr Thr Cys 290 295
300Ala Cys Gly Cys Cys Thr Thr Gly Gly Cys Thr Gly Cys Thr Ala
Ala305 310 315 320Ala Cys
Thr Ala Thr Thr Ala Cys Ala Gly Gly Ala Ala Ala Ala Cys
325 330 335Gly Ala Cys Ala Cys Ala Ala
Cys Thr Thr Thr Ala Gly Thr Gly Ala 340 345
350Ala Gly Ala Cys Thr Ala Ala Ala Gly Ala Ala Cys Thr Ala
Ala Thr 355 360 365Thr Ala Ala Ala
Ala Ala Thr Thr Ala Thr Ala Thr Thr Ala Cys Cys 370
375 380Gly Cys Cys Ala Gly Ala Ala Thr Ala Ala Thr Gly
Gly Cys Thr Ala385 390 395
400Ala Gly Ala Gly Ala Cys Cys Ala Thr Thr Thr Gly Ala Cys Ala Ala
405 410 415Ala Ala Ala Ala Thr
Cys Cys Ala Ala Cys Thr Cys Thr Gly Cys Thr 420
425 430Cys Thr Thr Thr Thr Thr Ala Gly Gly Gly Cys Cys
Gly Thr Cys Gly 435 440 445Gly Cys
Gly Ala Gly Gly Gly Thr Ala Ala Cys Gly Cys Ala Cys Ala 450
455 460Ala Thr Thr Gly Gly Thr Ala Gly Cys Cys Ala
Thr Thr Thr Thr Cys465 470 475
480Gly Gly Thr Gly Gly Thr Cys Ala Ala Gly Gly Thr Ala Ala Cys Ala
485 490 495Cys Cys Gly Ala
Cys Gly Ala Cys Thr Ala Cys Thr Thr Thr Gly Ala 500
505 510Ala Gly Ala Ala Thr Thr Gly Cys Gly Thr Gly
Ala Thr Cys Thr Ala 515 520 525Thr
Ala Thr Cys Ala Ala Ala Cys Thr Thr Ala Thr Cys Ala Thr Gly 530
535 540Thr Cys Thr Thr Ala Gly Thr Gly Gly Gly
Ala Gly Ala Thr Thr Thr545 550 555
560Ala Ala Thr Cys Ala Ala Gly Thr Thr Cys Thr Cys Cys Gly Cys
Thr 565 570 575Gly Ala Ala
Ala Cys Thr Thr Thr Ala Ala Gly Thr Gly Ala Ala Cys 580
585 590Thr Gly Ala Thr Thr Ala Gly Ala Ala Cys
Thr Ala Cys Thr Thr Thr 595 600
605Ala Gly Ala Thr Gly Cys Thr Gly Ala Ala Ala Ala Ala Gly Thr Cys 610
615 620Thr Thr Thr Ala Cys Thr Cys Ala
Ala Gly Gly Thr Thr Thr Ala Ala625 630
635 640Ala Cys Ala Thr Ala Thr Thr Gly Gly Ala Ala Thr
Gly Gly Thr Thr 645 650
655Gly Gly Ala Gly Ala Ala Cys Cys Cys Thr Thr Cys Ala Ala Ala Thr
660 665 670Ala Cys Cys Cys Cys Ala
Gly Ala Cys Ala Ala Gly Gly Ala Cys Thr 675 680
685Ala Thr Thr Thr Ala Cys Thr Thr Thr Cys Cys Ala Thr Thr
Cys Cys 690 695 700Ala Ala Thr Thr Thr
Cys Ala Thr Gly Cys Cys Cys Cys Thr Thr Ala705 710
715 720Ala Thr Thr Gly Gly Thr Gly Thr Cys Ala
Thr Thr Cys Ala Ala Thr 725 730
735Thr Gly Gly Cys Thr Cys Ala Cys Thr Ala Cys Gly Thr Ala Gly Thr
740 745 750Thr Ala Cys Thr Gly
Cys Cys Ala Ala Gly Cys Thr Thr Thr Thr Gly 755
760 765Gly Gly Thr Thr Thr Cys Ala Cys Thr Cys Cys Ala
Gly Gly Thr Gly 770 775 780Ala Gly Thr
Thr Ala Ala Gly Ala Thr Cys Thr Thr Ala Cys Thr Thr785
790 795 800Ala Ala Ala Ala Gly Gly Thr
Gly Cys Thr Ala Cys Ala Gly Gly Thr 805
810 815Cys Ala Cys Thr Cys Thr Cys Ala Ala Gly Gly Thr
Thr Thr Gly Gly 820 825 830Thr
Thr Ala Cys Thr Gly Cys Thr Gly Thr Cys Gly Cys Cys Ala Thr 835
840 845Ala Gly Cys Thr Gly Ala Gly Ala Cys
Gly Gly Ala Thr Thr Cys Cys 850 855
860Thr Gly Gly Gly Ala Ala Thr Cys Cys Thr Thr Cys Thr Thr Cys Gly865
870 875 880Thr Cys Thr Cys
Cys Gly Thr Ala Ala Gly Ala Ala Ala Ala Gly Cys 885
890 895Ala Ala Thr Thr Ala Cys Thr Gly Thr Ala
Thr Thr Ala Thr Thr Cys 900 905
910Thr Thr Cys Ala Thr Cys Gly Gly Thr Gly Thr Thr Cys Gly Thr Thr
915 920 925Gly Thr Thr Ala Cys Gly Ala
Ala Gly Cys Ala Thr Ala Cys Cys Cys 930 935
940Ala Ala Ala Cys Ala Cys Thr Thr Cys Cys Cys Thr Ala Cys Cys
Ala945 950 955 960Cys Cys
Ala Thr Cys Cys Ala Thr Cys Thr Thr Gly Gly Ala Ala Gly
965 970 975Ala Thr Thr Cys Cys Thr Thr
Gly Gly Ala Ala Ala Ala Cys Ala Ala 980 985
990Thr Gly Ala Ala Gly Gly Thr Gly Thr Thr Cys Cys Ala Thr
Cys Thr 995 1000 1005Cys Cys Ala
Ala Thr Gly Thr Thr Gly Thr Cys Cys Ala Thr Thr 1010
1015 1020Thr Cys Cys Ala Ala Thr Cys Thr Ala Ala Cys
Thr Cys Ala Ala 1025 1030 1035Gly Ala
Ala Cys Ala Ala Gly Thr Thr Cys Ala Ala Gly Ala Cys 1040
1045 1050Thr Ala Thr Gly Thr Ala Ala Ala Thr Ala
Ala Gly Ala Cys Thr 1055 1060 1065Ala
Ala Cys Thr Cys Thr Cys Ala Thr Thr Thr Gly Cys Cys Ala 1070
1075 1080Gly Cys Thr Gly Gly Thr Ala Ala Ala
Cys Ala Ala Gly Thr Thr 1085 1090
1095Gly Ala Ala Ala Thr Thr Thr Cys Thr Cys Thr Ala Gly Thr Cys
1100 1105 1110Ala Ala Thr Gly Gly Thr
Gly Cys Gly Ala Ala Gly Ala Ala Thr 1115 1120
1125Cys Thr Ala Gly Thr Cys Gly Thr Ala Thr Cys Gly Gly Gly
Cys 1130 1135 1140Cys Cys Ala Cys Cys
Ala Cys Ala Ala Thr Cys Ala Thr Thr Ala 1145 1150
1155Thr Ala Thr Gly Gly Thr Thr Thr Ala Ala Ala Cys Thr
Thr Gly 1160 1165 1170Ala Cys Thr Thr
Thr Ala Ala Gly Ala Ala Ala Gly Gly Cys Cys 1175
1180 1185Ala Ala Gly Gly Cys Cys Cys Cys Ala Thr Cys
Thr Gly Gly Ala 1190 1195 1200Cys Thr
Gly Gly Ala Thr Cys Ala Ala Thr Cys Ala Ala Gly Ala 1205
1210 1215Ala Thr Cys Cys Cys Ala Thr Thr Cys Ala
Gly Cys Gly Ala Ala 1220 1225 1230Ala
Gly Ala Ala Ala Ala Thr Thr Gly Ala Ala Gly Thr Thr Cys 1235
1240 1245Thr Cys Cys Ala Ala Thr Ala Gly Gly
Thr Thr Cys Thr Thr Ala 1250 1255
1260Cys Cys Thr Gly Thr Thr Gly Cys Ala Thr Cys Ala Cys Cys Ala
1265 1270 1275Thr Thr Cys Cys Ala Thr
Thr Cys Cys Cys Ala Thr Cys Thr Ala 1280 1285
1290Thr Thr Gly Gly Thr Thr Cys Cys Ala Gly Cys Thr Thr Cys
Ala 1295 1300 1305Gly Ala Thr Thr Thr
Gly Ala Thr Thr Ala Ala Cys Ala Ala Ala 1310 1315
1320Gly Ala Cys Thr Thr Ala Gly Thr Cys Ala Ala Ala Ala
Ala Cys 1325 1330 1335Ala Ala Thr Gly
Thr Cys Ala Gly Cys Thr Thr Thr Ala Ala Cys 1340
1345 1350Gly Cys Thr Ala Ala Ala Gly Ala Thr Ala Thr
Thr Cys Ala Ala 1355 1360 1365Ala Thr
Cys Cys Cys Cys Gly Thr Thr Thr Ala Cys Gly Ala Cys 1370
1375 1380Ala Cys Thr Thr Thr Thr Gly Ala Thr Gly
Gly Thr Thr Cys Ala 1385 1390 1395Gly
Ala Thr Cys Thr Ala Ala Gly Ala Gly Thr Cys Cys Thr Thr 1400
1405 1410Thr Cys Ala Gly Gly Thr Thr Cys Cys
Ala Thr Thr Thr Cys Cys 1415 1420
1425Gly Ala Gly Ala Gly Ala Ala Thr Cys Gly Thr Cys Gly Ala Cys
1430 1435 1440Thr Gly Cys Ala Thr Cys
Ala Thr Thr Ala Gly Ala Thr Thr Ala 1445 1450
1455Cys Cys Thr Gly Thr Cys Ala Ala Ala Thr Gly Gly Gly Ala
Ala 1460 1465 1470Ala Cys Thr Ala Cys
Thr Ala Cys Ala Cys Ala Ala Thr Thr Cys 1475 1480
1485Ala Ala Ala Gly Cys Cys Ala Cys Cys Cys Ala Cys Ala
Thr Ala 1490 1495 1500Thr Thr Ala Gly
Ala Cys Thr Thr Thr Gly Gly Thr Cys Cys Ala 1505
1510 1515Gly Gly Thr Gly Gly Ala Gly Cys Thr Thr Cys
Cys Gly Gly Thr 1520 1525 1530Thr Thr
Ala Gly Gly Thr Gly Thr Thr Thr Thr Ala Ala Cys Cys 1535
1540 1545Cys Ala Thr Cys Gly Thr Ala Ala Thr Ala
Ala Ala Gly Ala Thr 1550 1555 1560Gly
Gly Thr Ala Cys Thr Gly Gly Thr Gly Thr Thr Cys Gly Thr 1565
1570 1575Gly Thr Thr Ala Thr Cys Gly Thr Thr
Gly Cys Cys Gly Gly Thr 1580 1585
1590Ala Cys Thr Cys Thr Cys Gly Ala Cys Ala Thr Thr Ala Ala Cys
1595 1600 1605Cys Cys Ala Gly Ala Thr
Gly Ala Thr Gly Ala Thr Thr Ala Cys 1610 1615
1620Gly Gly Ala Thr Thr Cys Ala Ala Gly Cys Ala Ala Gly Ala
Ala 1625 1630 1635Ala Thr Cys Thr Thr
Thr Gly Ala Thr Gly Thr Thr Ala Cys Thr 1640 1645
165061950DNASaccharomyces cerevisiae 6ttggaaagag ttattgtagt
taccggtttt gctgaagtcg gcccatgggg ttcggccaga 60acaagatggg aaatggaagc
ttttggtgaa ttttcgttgg aaggttgcgt tgaaatggcc 120tggattatgg gcttcatttc
ataccataac ggtaatttga agggtcgtcc atacactggt 180tgggttgatt ccaaaacaaa
agaaccagtt gatgacaagg acgttaaggc caagtatgaa 240acatcaatcc tagaacacag
tggtatcaga ttgatcgaac cagagttatt caatggttac 300aacccagaaa agaaggaaat
gattcaagaa gtcattgtcg aagaagactt ggaaccattt 360gaggcttcga aggaaactgc
cgaacaattt aaacaccaac atggtgacaa agtggatatc 420ttcgaaatcc cagaaacagg
agagtactct gttaagttac taaagggtgc cactttatac 480attccaaagg ctttgagatt
tgaccgtttg gttgcaggtc aaattccaac tggttggaat 540gctaagactt atggtatctc
tgatgatatc atttctcagg ttgacccaat cacattattc 600gttttggtct ctgttgtgga
agcatttatt gcatctggta tcaccgaccc atacgaaatg 660tacaaatacg tacatgtttc
tgaggttggt aactgttctg gttctggtat gggtggtgtt 720tctgccttac gtggtatgtt
taaggaccgt ttcaaggatg agcctgtcca aaatgatatt 780ttacaagaat catttatcaa
caccatgtcc gcttgggtta atatgttgtt gatttcctca 840tctggtccaa tcaagacacc
tgttggtgcc tgtgccacat ccgtggaatc tgttgacatt 900ggtgtagaaa ccatcttgtc
tggtaaggct agaatctgta ttgtcggtgg ttacgatgat 960ttccaagaag aaggctcctt
tgagttcggt aacatgaagg ccacttccaa cactttggaa 1020gaatttgaac atggtcgtac
cccagcggaa atgtccagac ctgccaccac tacccgtaac 1080ggttttatgg aagctcaagg
tgctggtatt caaatcatca tgcaagctga tttagctttg 1140aagatgggtg tgccaattta
cggtattgtt gccatggctg ctaccgccac cgataagatt 1200ggtagatctg tgccagctcc
aggtaagggt attttaacca ctgctcgtga acaccactcc 1260agtgttaagt atgcttcacc
aaacttgaac atgaagtaca gaaagcgcca attggttact 1320cgtgaagctc agattaaaga
ttgggtagaa aacgaattgg aagctttgaa gttggaggcc 1380gaagaaattc caagcgaaga
ccaaaacgag ttcttacttg aacgtaccag agaaatccac 1440aacgaagctg aaagtcaatt
gagagctgca caacaacaat ggggtaacga cttctacaag 1500agggacccac gtattgctcc
attgagagga gcactggcta cttacggttt aactattgat 1560gacttgggtg tcgcttcatt
ccacggtaca tccacaaagg ctaatgacaa gaacgaatct 1620gccacaatta atgaaatgat
gaagcatttg ggtagatctg aaggtaatcc cgtcattggt 1680gttttccaaa agttcttgac
tggtcatcca aagggtgctg ctggtgcatg gatgatgaat 1740ggtgctttgc aaattctaaa
cagtggtatt attccaggta accgtaacgc tgataacgtg 1800gataagatct tggagcaatt
tgaatacgtc ttgtacccat ccaagacttt aaagaccgac 1860ggtgtcagag ccgtgtccat
cacttctttc ggttttggtc aaaagggtgg tcaagctatt 1920gtggttcatc cagactactt
atacggtgct 1950745DNAArtificial
SequencePrimer 7ttcttcgctg gtgttcgttg ttacgaagca tacccaaaca cttcc
45845DNAArtificial SequencePrimer 8acaccagcga agaataatac
agtaattgct tttcttacgg agacg 45942DNAArtificial
SequencePrimer 9agttgtgttc tacaaaggta tgactatgca agttgctgtt cc
421049DNAArtificial SequencePrimer 10catagtcata cctttgtaga
acacaacttc aactaaagat tcgatagac 491135DNAArtificial
SequencePrimer 11tctggttctt ctatgggtgg tgtttctgcc ttacg
351243DNAArtificial SequencePrimer 12catagaagaa ccagaacagt
taccaacctc agaaacatgt acg 431335DNAArtificial
SequencePrimer 13tctggttctt cttggggtgg tgtttctgcc ttacg
351443DNAArtificial SequencePrimer 14ccaagaagaa ccagaacagt
taccaacctc agaaacatgt acg 431535DNAArtificial
SequencePrimer 15attttacaag aatcatatat caacaccatg tccgc
351631DNAArtificial SequencePrimer 16tgattcttgt aaaatatcat
tttggacagg c 311751DNAArtificial
SequencePrimer 17tcttgtaaaa tagcattttg gacaggctca tccttgaaac ggtccttaaa c
511855DNAArtificial SequencePrimer 18cctgtccaaa atgctatttt
acaagaatca tttatcaaca ccatgtccgc ttggg 551951DNAArtificial
SequencePrimer 19tcttgtaaaa tatcagcttg gacaggctca tccttgaaac ggtccttaaa c
512055DNAArtificial SequencePrimer 20cctgtccaag ctgatatttt
acaagaatca tttatcaaca ccatgtccgc ttggg 552155DNAArtificial
SequencePrimer 21gaataatgtg attgggtcaa ccgcagaaat gatatcatca gagataccat
aagtc 552248DNAArtificial SequencePrimer 22ctgcggttga cccaatcaca
ttattcgttt tggtctctgt tgtggaag 482355DNAArtificial
SequencePrimer 23gaataatgtg attgggtcaa cctcagaaat gatatcatca gagataccat
aagtc 552448DNAArtificial SequencePrimer 24ctgaggttga cccaatcaca
ttattcgttt tggtctctgt tgtggaag 482551DNAArtificial
SequencePrimer 25tcttgtaaaa tatcatcttg gacaggctca tccttgaaac ggtccttaaa c
512655DNAArtificial SequencePrimer 26cctgtccaag atgatatttt
acaagaatca tttatcaaca ccatgtccgc ttggg 552767DNAArtificial
SequencePrimer 27cagtgaattg taatacgact cactataggg cgaattggag ctggcaaaac
gtaggggcaa 60acaaacg
672864DNAArtificial SequencePrimer 28gcaaaatatg agctaattct
tgctcaactt ccggcttcat tgtgtattac gatatagtta 60atag
642971DNAArtificial
SequencePrimer 29gcatacaatc aactatcaac tattaactat atcgtaatac acaatgaagc
cggaagttga 60gcaagaatta g
713021DNAArtificial SequencePrimer 30cacacaggaa acagctatga c
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