Patent application title: GENETICALLY MODIFIED YEASTS AND FERMENTATION PROCESSES USING GENETICALLY MODIFIED YEASTS
Inventors:
IPC8 Class: AC12P714FI
USPC Class:
1 1
Class name:
Publication date: 2020-11-19
Patent application number: 20200362371
Abstract:
The present invention relates to a genetically engineered yeast capable
of manufacturing a fermentation product using sucrose as a fermentation
substrate, and fermentation processes using such a yeast. The yeast has
an exogenous invertase gene and has a deletion or disruption of the PDC
activity gene. Accordingly, the yeast is useful for manufacturing
fermentation products other than ethanol from fermentation substrates
containing sucrose.Claims:
1-25. (canceled)
26. A process for manufacturing a fermentation product comprising: fermenting a substrate using a yeast, wherein the substrate comprises sucrose and the yeast comprises an exogenous invertase gene.
27. The process of claim 26, wherein the yeast is a yeast of the Issatchenkia orientalis/P. fermentans clade.
28. The process of claim 27, wherein the yeast is Issatchenkia orientalis.
29. The process of claim 26, wherein the yeast is PDC-negative.
30. The process of claim 26, wherein the process is microaerobic.
31. The process of claim 26, wherein the volumetric oxygen uptake rate (OUR) is 0.5 to 40 mmol O.sub.2/(Lh).
32-34. (canceled)
35. The process of claim 26, wherein the specific OUR is 0.2 to 13 mmol O.sub.2/(g cell dry weighth).
36-47. (canceled)
48. The process of claim 26, wherein the fermentation substrate comprises sucrose and glucose.
49. The process of claim 26, wherein the fermentation substrate comprises sucrose and hydrozylates of starch.
50. The process of claim 26, wherein the fermentation substrate comprises sucrose and xylose.
51. The process of claim 26, wherein the fermentation substrate comprises sucrose and lignocellulosic hydrozylates.
52. The process of claim 26, wherein the fermentation substrate comprises sucrose, glucose, and xylose.
53. The process of claim 26, wherein the process has a ratio of invertase activity to glucose consumption rate of less than 95.
54-55. (canceled)
56. The process of claim 26, wherein the process has a ratio of invertase activity to glucose consumption rate of at least 0.95.
57. (canceled)
58. The process of claim 26, wherein the fermentation yield is at least 55 percent.
59-61. (canceled)
62. The process of claim 26, wherein the final titer is at least 30 g/liter.
63. The process of claim 26, wherein the final titer is at least 80 g/liter.
64. (canceled)
65. The process of claim 26, wherein the fermentation product is selected from the group consisting of: lactic acid, citric acid, malonic acid, hydroxy butyric acid, adipic acid, lysine, keto-glutaric acid, glutaric acid, 3-hydroxy-proprionic acid, succinic acid, malic acid, fumaric acid, itaconic acid, muconic acid, methacrylic acid, and acetic acid and derivatives thereof and salts thereof.
66-68. (canceled)
69. The process of claim 26, wherein the invertase activity is in the range of about 2.5 to 50 (g glucose released/(g CDW*h)).
70. The process of claim 26, wherein the ratio of invertase activity to glucose consumption rate (or glucose capacity) is in the range of about 0.5 to 25.
71. (canceled)
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 62/259,531, filed Nov. 24, 2015, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] The entire contents of the ASCII text file entitled "N003 16SEQID2.txt," created on Nov. 22, 2016, and having a size of 82 kilobytes is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Industrial yeast fermentation processes often use glucose-based substrates in regions of the world where such substrates are readily available. For example, glucose syrup made from corn starch is commonly used in fermentation processes in the United States. However, in some regions, sucrose substrates are more readily available and/or more economical for use in fermentation processes, or it is desirable to use such sucrose substrates as a supplement to glucose substrates.
SUMMARY OF THE INVENTION
[0004] Described herein are genetically engineered yeasts useful for manufacturing fermentation products and fermentation processes based on the use of such yeasts. In one aspect, the present invention relates to engineering yeasts to use sucrose as a fermentation substrate from host yeasts that are incapable of using sucrose or are inefficient at using sucrose as a fermentation substrate. Accordingly, the yeasts of the present invention have a functional invertase gene. In one aspect, the yeasts are engineered to include promoters that are associated with an optimized expression of invertase.
[0005] In one aspect, the genetically engineered yeast comprises a yeast capable of producing a fermentation product at a production rate of at least 1.0 grams/liter-hour (g L.sup.-1 h.sup.-1), wherein the genetically engineered yeast has a functional invertase gene and has a deletion or disruption of the pyruvate decarboxylase (PDC) gene. In some embodiments, the yeast is capable of producing a fermentation product at a fermentation production rate of at least 1.5 g L.sup.-1 h.sup.-1 or at least 2.0 g L.sup.-1 h.sup.-1. In some embodiments, the yeast is capable of producing a fermentation product at a pathway fermentation yield of at least 55 percent, at least 65 percent, at least 70 percent, or at least 75 percent. In some embodiments, the yeast is capable of producing a fermentation product at a final titer of at least 30 g/liter, at least 80 g/liter, or at least 100 g/liter. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity of less than 95, less than 30, or less than 20. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity of at least 0.95 or at least 10. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity of at least 2.5, 3, or 5.
[0006] In another aspect, the genetically engineered yeast capable of manufacturing a fermentation product is a yeast of the I. orientalis/P. fermentans clade having a gene encoding a functional invertase. In one embodiment, such a yeast is PDC-negative. In one embodiment, the yeast is I. orientalis.
[0007] In some embodiments, the yeast is Crabtree-negative. In some embodiments, the functional invertase gene is selected from the group consisting of SEQ ID NO: 6; SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17. In some embodiments, the yeast includes an exogenous or artificial promoter for the functional invertase gene. In some embodiments, the promoter is selected from the group consisting of Pyruvate decarboxylase, Glyceraldehyde-3-phosphate dehydrogenase, Translational elongation factor, Transaldolase, RPL16B, 3-phosphoglycerate kinase, and Enolase. In some embodiments, the yeast is capable of manufacturing any of the following fermentation products: lactic acid, citric acid, malonic acid, hydroxy butyric acid, adipic acid, lysine, keto-glutaric acid, glutaric acid, 3-hydroxy-proprionic acid, succinic acid, malic acid, fumaric acid, itaconic acid, muconic acid, methacrylic acid, or acetic acid, or any derivatives thereof, any salts thereof, or any combinations thereof.
[0008] In one aspect, the process is a process for manufacturing a fermentation product comprising fermenting a substrate using any of the genetically engineered yeasts described herein. In one aspect, the process is a process for manufacturing a fermentation product comprising: fermenting a substrate using a yeast, wherein the substrate includes sucrose and the yeast includes an exogenous invertase gene.
[0009] In some embodiments, the process is microaerobic. In some embodiments, the volumetric oxygen uptake rate (OUR) is 0.5 to 40 mmol 02/(L-h), 1 to 30 mmol 02/(L-h), 3 to 20 mmol 02/(L-h), or 5 to 16 mmol 02/(L-h). In some embodiments, the specific OUR is 0.2 to 13 mmol 02/(g cell dry weight-h), 0.3 to 10 mmol 02/(g cell dry weight-h), 1 to 7 mmol 02/(g cell dry weight-h), or 2 to 6 mmol 02/(g cell dry weight-h).
[0010] In some embodiments, the fermentation cell concentration of the process is 1 to 10 g cell dry weight/L, 2 to 8 g cell dry weight/L, or 2.5 to 6 g cell dry weight/L. In some embodiments, the pitch density of the process is 0.05 to 5 g cell dry weight/L, 0.05 to 4 g cell dry weight/L, or 0.05 to 2 g cell dry weight/L. In some embodiments, the fermentation temperature is in the range of 25 to 45.degree. C., in the range of 20 to 40.degree. C., or in the range of 33 to 38.degree. C. In some embodiments, the fermentation substrate of the process comprises sucrose, glucose, hydrozylates of starch, xylose, lignocellulosic hydrozylates, or any mixture or any combination thereof.
[0011] In some embodiments, the process has a ratio of invertase activity to glucose consumption rate of less than 95, of less than 30, or of less than 20. In some embodiments, the process has a ratio of invertase activity to glucose consumption rate of at least 0.95 or at least 10. In some embodiments, the fermentation yield of the process is at least 55 percent, at least 65 percent, at least 70 percent, or at least 75 percent. In some embodiments, the final titer is at least 30 g/liter, at least 80 g/liter, or at least 100 g/liter. In some embodiments, the fermentation product of the process is lactic acid, citric acid, malonic acid, hydroxy butyric acid, adipic acid, lysine, keto-glutaric acid, glutaric acid, 3-hydroxy-proprionic acid, succinic acid, malic acid, fumaric acid, itaconic acid, muconic acid, methacrylic acid, or acetic acid, or any derivatives thereof, any salts thereof, or any combinations thereof.
[0012] In some embodiments, the invertase gene in the yeast is an integrated functional exogenous invertase gene. In some embodiments, the invertase activity of the yeast or the yeast in the process is at least 1, 2, 2.5, 3, 4, 5, 6, 7, 8, or 9 (g glucose released/(g CDW*h)). In some embodiments, the invertase activity of the yeast or the yeast in the process is less than 10, 15, 20, 30, 40, or 50 (g glucose released/(g CDW*h). In some embodiments, the invertase activity of the yeast or process is in the range of about 2.5-50, 5-30, or 5-20 (g glucose released/(g CDW*h)). In some embodiments, the ratio of invertase activity to glucose consumption rate (or glucose capacity) of the yeast or process is in the range of about 0.5 to 25 or 1 to 20.
[0013] It is also to be understood that the elements or aspects of any embodiment of the processes, methods, or compositions described above can be applied to any other embodiment, as would be understood by a person skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The following detailed description of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
[0015] FIG. 1 is a graph showing sucrose (squares), glucose (diamonds), fructose (triangles), and succinate (circles) titers for an exemplary fermentation process using yeast strain 1-8.
[0016] FIG. 2 is a graph showing glucose (solid lines) and succinate (dashed lines) titer for exemplary fermentation processes using strain 1-5 (circles) and strain 1-8 (squares).
[0017] FIG. 3 is a graph showing glucose (solid lines) and succinate (dashed lines) titer for an exemplary fermentation process using strain 1-1 (squares), strain 1-2 (circles) and strain 1-4 (triangles)
DETAILED DESCRIPTION
[0018] It is to be understood that the figures and descriptions of the present invention provided herein have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating other elements found in the related field(s) of art. Those of ordinary skill in the art would recognize that other elements or steps may be desirable or required in implementing the present invention. However, because such elements or steps are well known in the art or do not facilitate a better understanding of the present invention, a discussion of such elements or steps is not provided herein.
Definitions
[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. As used herein, each of the following terms has the meaning associated with it as defined in this section.
Fermentation Process Definitions
[0020] As used herein, "inoculation" is defined as the point in time wherein a microorganism capable of producing a fermentation product is introduced into a fermentation medium. This is a term that is well known to those skilled in the art.
[0021] As used herein, "end of fermentation" is defined as the point in time where a fermentation process meets a predetermined criteria. The predetermined criteria can include any of the following: a predetermined time interval, exhaustion of the desired fraction of carbon source supplied, cessation of carbon source consumption, or cessation of fermentation product formation. In one embodiment, "end of fermentation" is defined as the point in time where harvesting of the bioproduct is started. As would be understood by a person skilled in the art, "end of fermentation" can refer to a point in time that is different depending on the scale and purpose of the fermentation process. For a large-scale production fermentation process, the "end of fermentation" is preferably the point at which harvesting of the bioproduct is started, i.e., after product formation has effectively stopped.
[0022] As used herein, "cell dry weight" refers to the concentration of dry cell mass present in a fermentation medium at the time of measurement, as measured in a fermentation sample. Cell dry weight is commonly expressed in units of grams/liter (g/L).
[0023] As used herein, "cell dry weight at inoculation" refers to the concentration of dry cell mass present in a fermentation medium immediately following inoculation, as measured in a fermentation sample. For fed-batch fermentations, the initial cell dry weight is calculated based on the final volume of fermentation medium. Measurement of dry cell weight is a method known to those skilled in the art. Cell dry weight at inoculation is commonly expressed in units of g/L.
[0024] As used herein, "cell dry weight at end of fermentation" refers to the concentration of dry cell mass present in a fermentation medium at the end of fermentation, as measured in a fermentation sample. Cell dry weight at end of fermentation is commonly expressed in units of g/L.
[0025] As used herein, "final titer" refers to the concentration of a substance in the fermentation broth at the end of fermentation. The final titer is commonly expressed in units of g/L.
[0026] As used herein, "initial titer" refers to the concentration of a substance present at inoculation. The initial titer is commonly expressed in units of g/L.
[0027] As used herein, "batch time" refers to the amount of time that has elapsed between the inoculation and the end of fermentation. The batch time is commonly expressed in units of hours (h).
[0028] As used herein, "sugar consumption rate" for a batch process refers to the difference between the initial titer of a sugar present in the fermentation broth and the final titer of the same sugar (initial titer minus final titer) divided by the batch time. The sugar consumption rate is commonly expressed in units of grams per liter-hour (g L.sup.-1 h.sup.-1, which can also be abbreviated as (g/(L*h))). When applied to a continuous or semi-continuous process, the "sugar consumption rate" is determined using methods known in the art.
[0029] As used herein, the "specific sugar consumption rate" for a batch process refers to the sugar consumption rate divided by the cell dry weight at the end of fermentation. The specific sugar consumption rate is commonly expressed in units of (g sugar) (g cells).sup.-1 h.sup.-1. When applied to a continuous or semi-continuous process, the "specific sugar consumption rate" is determined using methods known in the art.
[0030] The sugar consumption rate and specific sugar consumption rate may be applied to specific sugars such as, for instance, glucose or sucrose. In these cases, one may refer to a glucose consumption rate, specific glucose consumption rate, sucrose consumption rate, or specific sucrose consumption rate.
[0031] As used herein, "fermentation production rate" for a batch process refers to the final titer minus initial titer of fermentation product (final titer minus initial titer) divided by the batch time. The production rate is commonly expressed in units of grams per liter-hour (g L.sup.-1 h.sup.-1). When applied to a continuous or semi-continuous process, the "fermentation production rate" is determined using methods known in the art.
[0032] As used herein, the "specific production rate" refers to the fermentation production rate divided by the cell dry weight at the end of fermentation. The specific production rate is commonly expressed in units of (g product) (g cells).sup.-1 h.sup.-1. When applied to a continuous or semi-continuous process, the "specific production rate" is determined using methods known in the art.
[0033] As used herein, "product yield" of a fermentation product refers to a ratio of two quantities: a) mass of product (e.g., succinate) produced in the course of the fermentation (numerator) b) the mass of carbon source added to the fermentation (denominator). The product yield as a percentage is commonly expressed in units of gram per gram (g/g) times 100. Particular note should be taken that product yield is calculated as a ratio of masses. The mass of fermentation product produced should account for the mass of fermentation product present in the fermentation medium at the end of the batch, as well as the mass of any fermentation product harvested during the course of the batch, less the mass of fermentation product present at the start of batch, and further less the mass of any fermentation product added during the course of the batch. The mass of carbon source added to the batch should include the mass of all carbon source(s) present in the fermenter at the start of the batch in addition to the mass of any carbon source(s) added during the course of the batch.
[0034] As used herein, "oxygen uptake rate" ("OUR") refers to the volumetric rate at which oxygen is consumed during a fermentation. Inlet and outlet oxygen concentrations can be measured with exhaust gas analysis, for instance by mass spectrometers. OUR can be calculated by one of ordinary skill in the relevant arts using the Direct Method described in Bioreaction Engineering Principles 2nd Edition, 2003, Kluwer Academic/Plenum Publishers, p. 449, equation 1. It is commonly measured in units of (mmol o2) L.sup.-1 h.sup.-1.
[0035] As used herein, "specific oxygen uptake rate" refers to the specific rate at which oxygen is consumed during a fermentation. It is calculated as the ratio of the OUR to the measured cell dry weight. It is commonly measured in units of mmol o2 (g cell dry weight).sup.-1 h.sup.-1.
[0036] As used herein, the term "microaerobic" refers to fermentation aeration conditions that are intermediate between fully aerobic and anaerobic conditions. Under microaerobic conditions, oxygen is supplied to the fermentation. Further, the oxygen is supplied at a rate such that the dissolved oxygen concentration is predominantly maintained below 5% of the saturation concentration of oxygen in the fermentation medium under air at atmospheric pressure. Under microaerobic conditions, the oxygen uptake rate is typically between 0.1 (mmol 0.sub.2) L.sup.-1 h.sup.-1 and 40 (mmol 0.sub.2) L.sup.-1 h.sup.-1
Yeast Characteristics Definitions
[0037] As used herein, the term "Crabtree-negative" refers to a yeast cell having a Crabtree-negative phenotype, i.e., any yeast cell that does not exhibit the Crabtree effect. In one embodiment, the host cell of the present invention is a Crabtree-negative yeast. The Crabtree effect concerns the inhibition of synthesis of respiratory enzymes. The Crabtree effect is defined as the occurrence of fermentative metabolism under aerobic conditions as a result of the inhibition of oxygen consumption by a microorganism when cultured at high specific growth rates (long-term effect) or in the presence of high concentrations of glucose (short-term effect). Organisms with the Crabtree negative phenotype do not exhibit this effect, and are thus able to consume oxygen even in the presence of high concentrations of glucose or at high growth rates. Whether an organism is Crabtree positive or Crabtree negative can be determined by comparing the ratio of fermented glucose to respired glucose during cultivation under aerobic conditions, with a ratio of greater than 1 indicative of a Crabtree positive organism (e.g., see De Deken, R. H. (1965) J. gen. Microbiol., 44:149-156).
[0038] As used herein, "sugar capacity" refers to the rate at which a yeast consumes a sugar as measured according to the method titled "strain capacity evaluation" as described below. The sugar capacity refers to the difference between the initial titer of a sugar present in the fermentation broth and the titer of the same sugar at the end of the evaluation (initial titer minus end titer) divided by the batch time, further divided by the cell dry weight at the end of the evaluation. The sugar capacity is commonly expressed in units of (g sugar) (g cells).sup.-1 h.sup.-1. This assay can be used to measure the sugar capacity for a number of sugars such as glucose or sucrose, resulting in, for example, a measurement of "glucose capacity" or "sucrose capacity."
sugar capacity = [ sugar ] initial - [ sugar ] end of evaluation ( batch time ) .times. ( cell dry weight at end of evaluation ) ##EQU00001##
[0039] For example, in an evaluation that lasts 45 hours, with 140.0 g/L glucose present at inoculation, 1.0 g/L glucose present at the end of the evaluation, and 6.0 g/L cell dry weight of yeast present at the end of fermentation, the calculated glucose capacity is 0.51 g glucose g.sup.-1 cells h.sup.-.
[0040] As used herein, "product capacity" refers to the rate at which a yeast produces a fermentation product as measured according the method titled "strain capacity evaluation" as described below. The product capacity refers to the difference between the initial titer of a product present in the fermentation broth and the titer of the same product at the end of the evaluation (initial titer minus end titer) divided by the batch time, further divided by the cell dry weight at the end of the evaluation. The product capacity is commonly expressed in units of (g product) (g cells).sup.-1 h.sup.-1. This assay can be used to measure the product capacity for a number of products such as lactate or succinate, resulting in a measurement of, for example, "lactate capacity" or "succinate capacity."
product capcity = [ p r o duct ] end of evaluation - [ p r o duct ] i n itial ( batch time ) .times. ( cell dry weight at end of evaluation ) ##EQU00002##
[0041] For example, in an evaluation that lasts 45 hours, with 0.0 g/L succinate present at inoculation, 100.0 g/L succinate present at the end of the evaluation, and 6.0 g/L cell dry weight of yeast present at the end of fermentation, the calculated succinate capacity is 0.37 g glucose g.sup.-1 cells h.sup.-1.
[0042] As used herein, "ratio of invertase activity to glucose capacity" refers to the ratio of invertase activity of a yeast strain, as measured according to the "invertase activity evaluation" method described below, to the observed glucose capacity of the same strain, as measured according to the "strain capacity evaluation" method described below. The units of this parameter are (g glucose released from sucrose hydrolysis/(g cell dry weight*hour))/(g glucose consumed/(g cell dry weight*hour)).
[0043] In certain embodiments, the genetically modified yeast cells provided herein further comprise a deletion or disruption of one or more native genes. As used herein, the phrase "deletion or disruption" with regard to a native gene means that either the entire coding region of the gene is eliminated (deletion) or the coding region of the gene, its promoter, and/or its terminator region is modified (such as by deletion, insertion, or mutation) such that the gene no longer produces an active enzyme, produces a severely reduced quantity (at least 75% reduction, preferably at least 90% reduction) of an active enzyme, or produces an enzyme with severely reduced (at least 75% reduced, preferably at least 90% reduced) activity.
[0044] In certain embodiments, deletion or disruption of one or more native genes results in a deletion or disruption of one or more native metabolic pathways. The phrase "deletion or disruption" with regard to a metabolic pathway means that the pathway is either inoperative or else exhibits activity that is reduced by at least 75%, at least 85%, or at least 95% relative to the native pathway. In certain embodiments, deletion or disruption of a native metabolic pathway is accomplished by incorporating one or more genetic modifications that result in decreased expression of one or more native genes that reduce ethanol production.
[0045] In some embodiments, deletion or disruption of native genes can be accomplished by forced evolution, mutagenesis, or genetic engineering methods, followed by appropriate selection or screening to identify the desired mutants. In some embodiments, deletion or disruption of a native host cell gene can be coupled to the incorporation of one or more exogenous genes into the host cell, i.e., the exogenous genes can be incorporated using a gene expression integration construct that is also a deletion construct. In some embodiments, deletion or disruption can be accomplished using a deletion construct that does not contain an exogenous gene or by other methods known in the art.
[0046] In some embodiments, the modified yeast cells described herein have a deletion or disruption of one or more native genes encoding an enzyme involved in ethanol fermentation or consumption, including for example pyruvate decarboxylase (PDC, catalyzes the conversion of pyruvate to acetaldehyde and carbon dioxide). Such modifications decrease the ability of the yeast cell to produce ethanol, thereby maximizing fermentation product production. In some embodiments where the modified yeast cell is I. orientalis, the cells comprise a deletion or disruption of a PDC gene encoding the amino acid sequence of SEQ ID NO: 14 and/or a gene encoding an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14.
[0047] As used herein, the terms "PDC-negative" or "PDC-" refer to a yeast which has a deletion or disruption of the pyruvate decarboxylase (PDC) gene. As would be understood by a person skilled in the art, deletion or disruption of the PDC gene will eliminate or reduce expression of PDC enzyme, which is an enzyme necessary for the production of ethanol via fermentation. In one embodiment, the pyruvate decarboxylase activity of the yeast is less than 0.05 U/milligram of total protein when using the methods previously described by Michele M. Bianchi, Lorenza Tizzani, Monika Destruelle, Laura Frontal and Micheline Wesolows ki-Louvel, The `petite-negative` yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity. (1996) Molecular Microbiology, 19 (1): 27-36. Biomass used for the assay is grown in YP media with 2% glucose. The activity unit (U) is defined as the amount of activity required for the conversion of 1 micromole of substrate (in this example, NADH to NAD+) per minute.
[0048] The term "exogenous" as used herein with regard to genetic components means that the genetic component is present in a modified version of a microorganism, but is not present in the genome of a native form of the particular microorganism cell. In some embodiments, the exogenous genetic component can be a modified form of a component that was native to the cell, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically-derived component. For example, the K. lactis invertase gene is exogenous when introduced into I. orientalis.
[0049] Inspection of nucleic acid or amino acid sequences for two nucleic acids or two polypeptides will reveal sequence identity and similarities between the compared sequences. Sequence alignment and generation of sequence identity include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1, -2; Gap costs: Linear; Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX % (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX % identical or, equivalently, have XX % sequence identity to the reference sequence.
[0050] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 7 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 6, from 2 to 5, from 3 to 5, etc., as well as individual numbers within that range, for example, 1, 2, 3, 3.6, 4, 5, 5.8, 6, 7, and any whole and partial increments in between. This applies regardless of the breadth of the range.
DESCRIPTION
[0051] Described herein are genetically modified yeast strains useful for manufacturing a fermentation product and fermentation processes using these yeasts. The yeast strains are modified to include a functional exogenous invertase gene. Accordingly, in one embodiment, the present invention relates to a yeast strain useful for fermentation processes having sucrose as a substrate. The yeast strain is preferably PDC-negative, and therefore can be useful for manufacturing fermentation products other than ethanol, for example succinic acid. In one embodiment, the yeast is Crabtree negative.
[0052] As contemplated herein, sucrose-based fermentation processes would preferably use a yeast expressing the invertase enzyme. However, invertase expression is not native to many yeasts that are desirable for industrial fermentation processes. Feng et al., describe the relationship between the fermentation activity of Saccharomyces cerevisiae in high-sugar dough and sucrase activity (Modern Food Sci. and Tech., 2014, 30:131-135). However, S. cerevisiae is primarily used for the production of ethanol, i.e., it has pyruvate decarboxylase (PDC) activity, and it is less desirable for use in manufacturing many other types of industrial chemicals. As would be understood by a person of ordinary skill in the art, deletion or disruption of the PDC gene in S. cerevisiae is highly problematic. This deletion in S. cerevisiae results in the loss of the ability to grow on glucose, as well as causing an autotrophy for c2 compounds (Flikweert et al., Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae, FEMS Microbiol Lett 1999; 174(1):73-9).
Genetically Engineered Yeast
[0053] The genetically modified yeast of the present invention is made by performing one or more genetic modifications to a host yeast cell. In some embodiments, the host yeast cell lacks a native invertase. In some embodiments, the host yeast cell does not include a nucleic acid encoding a polypeptide with a sequence that has greater than 70% identity with SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the host yeast cell cannot grow on sucrose as a sole carbon source. In some embodiments, the host yeast cell has a maximum specific growth rate on (YNB+20 g/L glucose) media that exceeds 0.15 h.sup.-1 and a maximum specific growth rate on (YNB+20 g/L sucrose) media that is less than 0.05 h.sup.-1. In some embodiments, the host yeast is a Crabtree-negative yeast.
[0054] In some embodiments, the genetically modified yeast cells described herein belong to the genus Issatchenkia, and in some such embodiments the yeast cells are I. orientalis. When first characterized, the species I. orientalis was assigned the name Pichia kudriavzevii. I. orientalis yeasts have also been described in the art as C. krusei. Numerous additional synonyms for the species I. orientalis have been described (see Kurtzman and Fell, The Yeasts, a Taxonomic Study, Section 35, Issatchenkia Kudryavtsev, pp. 222-223 (1998), which is hereby incorporated by reference).
[0055] The I. orientalis/P. fermentans clade is the most terminal clade that contains at least the species I. orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida ethanolica, Pichia deserticola, P. membranifaciens, and P. fermentans. Members of the I. orientalis/P. fermentans clade are identified by analysis of the variable D1/D2 domain of the 26S ribosomal DNA of yeast species, using the method described by Kurtzman and Robnett in "Identification and Phylogeny of Ascomycetous Yeasts from Analysis of Nuclear Large Subunit (26S) Ribosomal DNA Partial Sequences," Antonie van Leeuwenhoek 73:331-371, 1998, which is hereby incorporated by reference (see especially p. 349). Analysis of the variable D1/D2 domain of the 26S ribosomal DNA from hundreds of ascomycetes has shown that the I. orientalis/P. fermentans clade contains very closely related species. Members of the I. orientalis/P. fermentans clade exhibit greater similarity in the variable D1/D2 domain of the 26S ribosomal DNA to other members of the clade than to yeast species outside of the clade. Therefore, other members of the I. orientalis/P. fermentans clade can be identified by comparison of the D1/D2 domains of their respective ribosomal DNA, and comparing to that of other members of the clade and closely related species outside of the clade, using Kurtzman and Robnett's methods.
[0056] As described herein, the present invention relates to genetically modified yeasts of the I. orientalis/P. fermentans clade, preferably I. orientalis. However, the present invention is not limited to using any specific yeast such as I. orientalis, and the host yeast cell can be any suitable yeast strain, as would be understood by a person skilled in the art. To genetically modify the yeast cell, a suitable locus is selected for gene integration. One of ordinary skill in the art would know how to select suitable loci in a yeast genome for gene integration. An example of a suitable locus for integration of exogenous genes in I. orientalis includes, but is not limited to, locus A, which is flanked by SEQ ID NO: 1 and SEQ ID NO: 2. Further, one of ordinary skill in the art would recognize how to use sequences to design PCR primers to verify correct gene integration at the chosen locus.
[0057] As contemplated herein, the genetically modified or engineered yeast of the present invention includes a functional exogenous invertase expression gene and has a deletion or disruption of the PDC gene. In one embodiment, the genetically modified yeast can include one or more additional exogenous integrated genes other than the integrated functional invertase expression gene. In one embodiment, the genetically modified yeast can include more than one functional invertase expression gene. In another embodiment, the genetically modified yeast can include a functional sucrase gene instead of, or in addition to, the invertase gene. For the purposes of this disclosure, an integrated gene does not include a gene maintained on a plasmid.
[0058] Exemplary invertase expression genes suitable for gene integration in a yeast strain include, but are not limited to: an invertase gene from K. lactis (KIIN V); S. cerevisiae (ScSUC2); Schizosaccharomyces pombe (invl); and Aspergillus niger (invA) also identified as SEQ ID NO: 6; SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17, respectively.
[0059] The genetically modified yeast of the present invention can also include exogenous or artificial promoters for the functional exogenous invertase expression gene or any other gene integrated into the yeast. One skilled in the art would know how to select and integrate suitable promoters into the host yeast cell. Examples of suitable promoters include, but are not limited to the promoters for the following I. orientalis genes: Pyruvate Decarboxylase (PDC), Glyceraldehyde-3-phosphate dehydrogenase (TDH3), Translational elongation factor (TEF), Transaldolase (TAL), RPL16B, 3-phosphoglycerate kinase (PGK), and Enolase (ENO).
[0060] In some embodiments, the integrated functional exogenous invertase expression may be associated with invertase activity which, once integrated into the host yeast cell, can be significantly greater than the desirable or optimal invertase activity. Greater than desired invertase activity can result in a less than optimal fermentation process. Greater than desired invertase activity can be problematic for a host cell and result in a reduction in the sugar consumption rate of the cell. While not wishing to be bound by theory, this reduction in sugar consumption rate can be due to the metabolic burden associated with producing large quantities of invertase protein, or can be due to other reasons that are not well understood.
[0061] Accordingly, the present invention also relates to the adjustment of invertase expression associated with the genetically modified yeast. Invertase expression in the genetically modified yeast can be optimized through one or more techniques known in the art. For example, in one embodiment, the amino acid sequence of invertase can be modified to reduce activity. In another embodiment, promoters associated with lower expression of invertase can be identified and integrated into the host yeast. However, the methods and compositions for optimizing invertase expression are not limited to those described herein, and can include any methods or compositions for adjusting or optimizing the invertase expression, as would be understood by a person skilled in the art.
[0062] In some embodiments, the yeast can be engineered for improved acetate consumption. Acetate consumption can be improved by overexpression of a gene encoding for an aldehyde dehydrogenase, or an acetyl-CoA synthase. In some embodiments, acetate consumption can be further improved by providing the cell with a greater pool of reducing equivalents to assist in the oxido-reduction of acetate to ethanol. One example of a genetic modification that can increase the pool of reducing equivalents is the deletion or disruption of a gene encoding a glycerol-3-phosphate dehydrogenase (GPD).
[0063] In some embodiments, the yeast can include heterologous expression of a transporter that can increase hexose uptake. An example of a transporter than can increase hexose uptake is Hxtl transporter of S. cerevisiae. One skilled in the art would recognize that yeasts are known to have other transporters capable of hexose uptake.
[0064] In some embodiments, the genetically engineered yeast of the present invention is capable of manufacturing a fermentation product other than ethanol. In some embodiments, the yeast is capable of producing a fermentation product at a production rate of at least 1.0 grams per liter-hour (g L.sup.-1 h.sup.-1), at least 1.5 g L.sup.-1 h.sup.-1, or at least 2.0 g L.sup.-1 h.sup.-1. In some embodiments, the yeast is capable of producing a fermentation product at a pathway fermentation yield of at least 55 percent, at least 65 percent, at least 70 percent, or at least 75 percent. In some embodiments, the yeast is capable of producing a fermentation product at a final titer of at least 30 g/liter, at least 80 g/liter, or at least 100 g/liter.
[0065] As contemplated herein, the genetically engineered yeast is capable of producing a fermentation product using sucrose as a fermentation substrate. The ratio of invertase activity to the rate of glucose consumption via fermentation can be optimized to maximize the manufacture of fermentation product. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity of less than 95, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 35, less than 30, less than 25 or less than 20. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity of at least 0.95 or at least 10. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 18. In some embodiments, the yeast has a ratio of invertase activity to glucose capacity in the range of 0.5 to 95, 0.5 to 30, 0.5 to 25, 0.5 to 20, or 1 to 20.
[0066] In some embodiments, the invertase activity of the yeast is at least 1, 2, 2.5, 3, 4, 5, 6, 7, 8, or 9 g glucose released/ (g CDW*h). In some embodiments, the invertase activity of the yeast is less than 10, 15, 20, 30, 40, or 50 g glucose released/(g CDW*h). In some embodiments, the invertase activity of the yeast is in the range of 1 to 50, 2.5 to 50, 2.5 to 25, 3 to 30, 5 to 30, 3 to 20, or 5 to 20.
[0067] The yeast can also be capable of producing a fermentation product using other fermentation substrates in addition to sucrose. In one embodiment, the yeast is capable of using a fermentation substrate that includes sucrose and glucose. In another embodiment, the yeast is capable of using a fermentation substrate that includes sucrose and xylose. In yet another embodiment, the yeast is capable of using a fermentation substrate that includes sucrose, glucose, and xylose. In some embodiments, the yeast is capable of using a fermentation substrate that includes hydrozylates, for example hydrozylates of starch or lignocellulosic hydrozylates. In some embodiments, the yeast is capable of using a fermentation substrate that includes any mixture or combination of sucrose, glucose, fructose, xylose, hydrozylates of starch, or lignocellulosic hydrozylates. As would be understood by a person skilled in the art, the yeast can be used with a fermentation substrate that does not include sucrose.
[0068] In one embodiment, the yeast of the present invention can include one or more inducible promoters. For example, the yeast may include a promoter capable of turning off invertase expression after most or all of the sucrose in the fermentation substrate has been hydrolyzed. As a further example, the yeast may contain a promoter that is capable of down regulating after the dissolved oxygen is reduced below a threshold.
Fermentation Processes
[0069] The present invention also relates to processes for manufacturing a fermentation product. The fermentation processes includes the step of fermenting a substrate using the genetically engineered yeasts described herein. The fermentation process can also include other steps, as would be understood by a person skilled in the art. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and isolating the fermentation product from the fermentation broth. In some embodiments, the fermentation process is a microaerobic process.
[0070] The fermentation processes of the present invention can be run using sucrose as a substrate, as a result of using genetically engineered yeasts having a functional invertase gene. The substrate of the fermentation process can also include other components in addition to sucrose. In one embodiment, the fermentation process substrate can also include glucose, xylose, fructose, hydrozylates of starch, lignocellulosic hydrozylates, or any combination thereof. As contemplated herein, the sucrose component of the substrate will be hydrolyzed into glucose and fructose via the activity of invertase and/or sucrase. Accordingly, in some embodiments, the fermentation substrate may not contain any sucrose because all of the sucrose may be hydrolyzed at some point during the process.
[0071] The fermentation process can be run under various conditions. In one embodiment, the fermentation temperature, i.e., the temperature of fermentation broth during processing, is ambient temperature. In some embodiments, the fermentation temperature is maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25 to 45.degree. C., 20 to 40.degree. C., or 33 to 38.degree. C. However, the fermentation temperature is not limited to any specific range recited herein.
[0072] The fermentation process can be run within certain oxygen uptake rate (OUR) ranges. In some embodiments, the volumetric OUR of the fermentation process can be in the range of 0.5 to 40, 1 to 30, 3 to 20, or 5 to 16 mmol 0 2/(L-h). In some embodiments, the specific OUR can be in the range of 0.2 to 13, 0.3 to 10, 1 to 7, or 2 to 6 mmol O{circumflex over ( )}g cell dry weight-h). However, the volumetric or specific OURs of the fermentation process are not limited to any specific rates or ranges recited herein.
[0073] The fermentation process can be run at various cell concentrations. In some embodiments, the cell dry weight at the end of fermentation can be 1 to 20, 1 to 10, 2 to 8, or 2.5 to 6 g cell dry weight/L. Further, the pitch density or pitching rate of the fermentation process can vary. In some embodiments, the pitch density can be 0.05 to 5, 0.05 to 4, or 0.05 to 2 g cell dry weight/L.
[0074] In addition, the fermentation process can be associated with various characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and the ratio of invertase activity to glucose consumption rate. In some embodiments, these characteristics can be affected based on the selection of the yeast and/or genetic modification of the yeast used in the fermentation process. In some embodiments, these characteristics can be affected by adjusting the fermentation process conditions. In some embodiments, these characteristics can be adjusted via a combination of yeast selection or modification and the selection of fermentation process conditions.
[0075] In some embodiments, the fermentation production rate of the process is at least 1.0, at least 1.5, or at least 2.0 g L.sup.-1 h.sup.-1. In some embodiments, the pathway fermentation yield of the process is at least 55 percent, at least 65 percent, at least 70 percent, or at least 75 percent. In some embodiments, the final titer of the process is at least 30, at least 80, or at least 100 g/liter. In some embodiments, the process has a ratio of invertase activity to glucose consumption rate of less than 95, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 35, less than 30, less than 25 or less than 20. In some embodiments, the process has a ratio of invertase activity to glucose consumption rate of at least 0.95 or at least 10. In some embodiments, the process has a ratio of invertase activity to glucose consumption rate of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 18. In some embodiments, the process has a ratio of invertase activity to glucose consumption rate in the range of 0.5 to 95, 0.5 to 30, 0.5 to 25, 0.5 to 20, or 1 to 20.
[0076] In some embodiments, the invertase activity of the process is at least 1, 2, 2.5, 3, 4, 5, 6, 7, 8, or 9 g glucose released/(g CDW*h). In some embodiments, the invertase activity of the process is less than 10, 15, 20, 30, 40, or 50 g glucose released/(g CDW*h). In some embodiments, the invertase activity of the process is in the range of 1 to 50, 2.5 to 50, 2.5 to 25, 3 to 30, 5 to 30, 3 to 20, or 5 to 20.
[0077] In some embodiments, the fermentation process can include sucrose as a substrate for only a portion of the process. For example, in one embodiment, the fermentation process can include the step of generating a yeast seed using sucrose as substrate, then running the full production batch with a hydrolysate, a hydrolysate supplemented with sucrose, or other substrate instead of sucrose. In one such embodiment, the fermentation process can be run as a sucrose-fed batch. Further, the fermentation process can be a batch process, continuous process, or semi-continuous process, as would be understood by a person skilled in the art.
Fermentation Products
[0078] The genetically engineered yeast of the present invention and the fermentation processes using the genetically engineered yeast can be used to manufacture a variety of compounds. Exemplary fermentation products that can be manufactured using the genetically engineered yeast include, but are not limited to: amino acids, organic acids, hydroxyl-organic acids, alcohols such as butanol, polyols, fatty acids, fatty acids such as methyl esters, monoacyl glycerides, diacyl glycerides, triacyl glycerides, and mixtures thereof. Exemplary organic acids or amino acids include lactic acid, citric acid, malonic acid, hydroxy butyric acid, adipic acid, lysine, keto-glutaric acid, glutaric acid, 3-hydroxy-proprionic acid, succinic acid, malic acid, fumaric acid, itaconic acid, muconic acid, methacrylic acid, and acetic acid and derivatives thereof and salts thereof. It is contemplated herein that isolation of the desired fermentation product produced from the fermentation process can be achieved via techniques well known to those skilled in the relevant art.
EXPERIMENTAL EXAMPLES
[0079] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
Evaluation Protocols
Strain Capacity Evaluation
[0080] The following protocol is used for evaluating the sugar capacity or product capacity of a yeast strain, as defined herein. Fermenters are inoculated with biomass grown in defined medium (adapted from Verduyn, et. al, 1992, Yeast 8, 501-517, see Tables 1, 3, and 4). Seeds are run in 250 mL baffled flasks (50 mL working volume) at 250 rpm and 30.degree. C. The contents of the flasks are harvested at approximately 16-24 hours incubation time. The cell density of the shake flask is measured and a volume of the shake flask broth is selected and inoculated into the fermenter such that the cell dry weight at inoculation is 0.1 g/L. Fermenter initial working volume is 1.5 L. Fermenter media is used as listed in Tables 2, 3, and 4. Sugar is provided by the addition of 140 g/l at the start of the batch (straight batch). pH is started at the ambient pH of the media (4-6) and is controlled at 3.0 with a combination of 28% NH.sub.4OH and 30% Ca(OH).sub.2. 3.8 g per 1.5 L media 28% NH4OH is used as initial pH control. Once this is exhausted, pH control is switched to Ca(OH)2 for the remainder of the batch. The fermenter systems are sparged at 0.24 slpm with a blend of pure CO2 and air to target 21-23% CO2 in the inlet gas stream. The fermentation is operated such that after the cells achieve a sufficient density, oxygen limitation is achieved and subsequently maintained throughout the rest of the fermentation (e.g., dissolved oxygen less than about 10%). Agitation rate is selected to achieve a peak oxygen uptake rate (OUR) in the fermentation of 21-22 mmol/L-h. The fermentation proceeds until the end of the evaluation which occurs when the sugar is reduced below 2 g/L or until the cessation of product formation, whichever occurs first. Samples are taken immediately after inoculation and at the end of the evaluation. These samples are used for cell dry weight determination as well as HPLC analysis for determination of sugar and product concentrations.
Invertase Activity Evaluation
[0081] The capability of a cell to convert sucrose to glucose and fructose is evaluated by the following protocol. The strains are taken from a fresh YPD plate and used to inoculate 50 mL of YPD liquid media. The culture is allowed to grow at 30.degree. C./250 rpm overnight (16 hours). Fresh cultures are inoculated to an OD6oo=1.0 in 50 mL of YPD liquid media and allowed to grow at 30.degree. C./250 rpm for 3 hours. The cells are harvested by centrifugation at 3,500 rpm for 4 minutes. The pellets are washed with 25 mL of water and centrifuged at 3,500 rpm for 4 minutes; this step is repeated 2 times. Washed cells are resuspended in 5 mL of water. 10 .mu.L of cell suspension is incubated with 40 .mu.L water, 250 .mu.L of 0.2 M sodium acetate, pH 4.9 and 125 of 0.5 M sucrose for 10 min at 37.degree. C. Samples are filtered through a 0.22 .mu.m filter. The glucose released is immediately measured on a YSI2950 (Xylem Inc.). The activity is expressed as grams of glucose released per gram of cell dry weight/hour. Assays are carried out in duplicate.
[0082] This assay is adapted from Silveira, M. C. F., Carvajal, E., Bon, E. P. S., Assay for in vivo yeast invertase activity using NaF (1996) Analytical Biochemistry, 238 (1), pp. 26-28, and Georis, I., Cassart, J.-P., Breunig, K. D., Vandenhaute, Glucose repression of the Kluyveromyces lactis invertase gene KIINV1 does not require Miglp (1999), Molecular and General Genetics 261(4-5):862-70.
Example 1: Genetically Modified Yeast Strains
Strain 1-1
[0083] Strain P-8b described by Rush et al. (Int'l. App. No. PCT/US2013/052069) is an evolved Issachenkia orientalis host strain in which both alleles of the URA3, PDC and GPD genes are deleted followed by the addition of diploid alleles of the following genes under control of heterologous promoters: I. orientalis PYC1, Schizosaccharomyces pombe MAE, Leshmania mexicana FRD, Rhizopus delemar MDH, and I. orientalis FUM (SEQ ID NO: 4). Strain 1-1 is created using the methods to create strain P-8b with the following change: 1) In Strain 1-1, the L. mexicana FRD gene of P-8b is replaced with the variant of the L. mexicana FRD gene of SEQ ID NO: 3.
Strain 1-1a
[0084] Strain 1-1 is grown for several rounds on 5-fluoroorotic acid (FOA) plates to identify a strain in which the URA3 marker has looped out. Resulting isolates are streaked for single colony isolation on YPD plates. A single colony is selected. Loss of the URA3 marker is verified by PCR. A PCR verified isolate is designated Strain 1-1a.
Strain 1-2
[0085] Strain 1-1a is transformed with SEQ ID NO: 5. SEQ ID NO: 5 contains: i) an expression cassette for the selectable marker gene URA from I. orientalis (IoURA) including a repeated portion of the URA promoter; ii) an expression cassette for an invertase from K. lactis (KIINV), encoding the amino acid sequence SEQ ID NO: 6 expressed by the PDC promoter SEQ ID NO: 7; and iii) flanking DNA for targeted chromosomal integration into integration locus A. Transformants are selected on ScD-Uracil plates. Resulting transformants are streaked for single colony isolation on ScD-Uracil plates. A single colony is selected. Correct integration of SEQ ID NO: 5 into the selected colony is verified by PCR. A PCR verified isolate is designated Strain 1-2.
Strain 1-3
[0086] Strain 1-1a is transformed with SEQ ID NO: 8. SEQ ID NO: 8 contains: i) an expression cassette for the selectable marker gene URA from I. orientalis (IoURA) including a repeated portion of the URA promoter; ii) an expression cassette for an invertase from K. lactis (KIINV), encoding the amino acid sequence SEQ ID NO: 6 expressed by the TAL promoter SEQ ID NO: 9; and iii) flanking DNA for targeted chromosomal integration into integration locus A. Transformants are selected on ScD-Uracil plates. Resulting transformants are streaked for single colony isolation on ScD-Uracil plates. A single colony is selected. Correct integration of SEQ ID NO: 8 into the selected colony is verified by PCR. A PCR verified isolate is designated Strain 1-3.
Strain 1-4
[0087] Strain 1-1a is transformed with SEQ ID NO: 10. SEQ ID NO: 10 contains: i) an expression cassette for the selectable marker gene URA from I. orientalis (IoURA) including a repeated portion of the URA promoter; ii) an expression cassette for an invertase from K. lactis (KIINV), encoding the amino acid sequence SEQ ID NO: 6 expressed by the RPL16B promoter SEQ ID NO: 11; and iii) flanking DNA for targeted chromosomal integration into integration locus A. Transformants are selected on ScD-Uracil plates. Resulting transformants are streaked for single colony isolation on ScD-Uracil plates. A single colony is selected. Correct integration of SEQ ID NO: 10 into the selected colony is verified by PCR. A PCR verified isolate is designated Strain 1-4.
Strain 1-5
[0088] Strain P-8b as described in the section titled "Strain 1-1" above is co-transformed with the integration fragments 6-1 and 6-2 listed in the second column of Table 3 in Rush et al. (Int'l. App. No. PCT/US20 13/052069). Integration fragments 6-1 and 6-2 target the E. coli transhydrogenase gene to the GPD locus. Successful integrants in each case are identified as blue colonies on selection plates with 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside and lacking uracil, and confirmed by PCR. A PCR verified isolate is designated Strain 1-5.
Strain 1-6
[0089] Strain 1-5 is transformed with the plasmid of SEQ ID NO: 12. SEQ ID NO: 12 contains: i) an expression cassette for the selectable marker gene invertase from S. cerevisiae (ScSUC2); and ii) an expression cassette for CRE recombinase gene (Cre) to recycle the selectable markers ScMEL5 & IoCYB2A. Transformants are selected on YNB plates containing 2% sucrose as sole carbon source and 32 .mu.g/mL x-alpha-gal which provides colorimetric indication of the absence of the ScMEL5 marker gene. Resulting transformants are streaked for single colony isolation on YPD containing 32 .mu.g/mL x-alpha-gal. A single white colony is selected. Loss of ScMEL5 and IoCYB2A from the selected white colony is verified by PCR. A PCR verified isolate is designated Strain 1-5a.
[0090] Strain 1-5a is grown for several rounds on 5-fluoroorotic acid (FOA) plates to identify a strain in which the URA3 marker has looped out. Resulting isolates are streaked for single colony isolation on YPD plates. A single colony is selected. Loss of the URA3 marker is verified by PCR. A PCR verified isolate is designated Strain 1-6.
Strain 1-7
[0091] Strain 1-6 is transformed with SEQ ID NO: 10. SEQ ID NO: 10 contains: i) an expression cassette for the selectable marker gene URA from I. orientalis (IoURA) including a repeated portion of the URA promoter; ii) an expression cassette for an invertase from K. lactis (KIINV), encoding the amino acid sequence SEQ ID NO: 6 expressed by the RPL16B promoter SEQ ID NO: 11; and iii) flanking DNA for targeted chromosomal integration into integration locus A. Transformants are selected on ScD-Uracil plates. Resulting transformants are streaked for single colony isolation on ScD-Uracil plates. A single colony is selected. Correct integration of SEQ ID NO: 10 into the selected colony is verified by PCR. A PCR verified isolate is designated Strain 1-7.
Strain 1-8
[0092] Strain 1-7 is transformed with SEQ ID NO: 13. SEQ ID NO: 13 contains: 1) an expression cassette for the selectable marker gene melibiase from S. cerevisiae (ScMEL5) flanked by LoxP sites; ii) an expression cassette for an invertase from K. lactis (KIINV), encoding the amino acid sequence SEQ ID NO: 6 expressed by the RPL16B promoter SEQ ID NO: 11; and iii) flanking DNA for targeted chromosomal integration into integration locus A. Transformants are selected on YNB plates containing 2% melibiose as sole carbon source and 32 .mu.g/mL x-alpha-gal which provides colorimetric indication of the presence of the ScMEL5 marker gene. Resulting transformants are streaked for single colony isolation on YPD containing 32 .mu.g/mL x-alpha-gal. A single blue colony is selected. Correct integration of SEQ ID NO: 13 into the selected blue colony is verified by PCR. A PCR verified isolate is designated Strain 1-8.
Example 2: Fermentation Using Genetically Modified Yeast Strains
[0093] This Example demonstrates the capability of the recombinant yeast strains having an exogenous invertase activity gene described above to convert sucrose to glucose and fructose, and subsequently and/or concurrently convert glucose to a fermentation product such as succinic acid.
Fermentation Conditions for Strains 1-1, 1-2, 1-3, and 1-4
[0094] The yeast strains 1-1, 1-2, 1-3, and 1-4, are run in fermenters to test succinic acid production. Fermenters are inoculated with biomass grown in defined medium (adapted from Verduyn, et. al, 1992, Yeast 8, 501-517, see Tables 1, 3, and 4). Seeds are run in 250 mL baffled flasks (50 mL working volume) at 250 rpm and 30.degree. C. The contents of the flasks are harvested at approximately 16-24 hours incubation time with 10% v/v inoculum used to start fermenters. Fermenter initial working volume is 1.5 L. The cell dry weight at inoculation is found in Table 5. Fermenter media is outlined in Tables 2, 3, and 4. Glucose or sucrose is added to achieve a concentration of 140 g/L at the start of the batch (straight batch). pH is started at the ambient pH of the media (4-6) and is allowed to drop to pH 3.0, after which it is controlled at 3.0 for the remainder of the batch with a combination of 28% NH.sub.4OH and 30% Ca(OH)2. 3.8 g per 1.5 L media 28% NH4OH is used as initial pH control. Once this is exhausted, pH control is switched to Ca(OH)2 for the remainder of the batch. The fermenter systems are sparged at 0.24 slpm with a blend of pure CO2 and air to target 21-23% CO2 in the inlet gas stream. These fermentations are operated such that after the cells achieve a sufficient density, oxygen limitation is achieved and subsequently maintained throughout the rest of the fermentation (e.g., dissolved oxygen less than about 10%). Agitation rate is selected to achieve a peak oxygen uptake rate (OUR) in the fermentations target 21-22 mmol/L-h.
Fermentation Conditions for Strains 1-5 and 1-8
[0095] The yeast strains 1-5 and 1-8 are run in fermenters to test succinic acid production. Fermenters are inoculated with biomass grown in defined medium (adapted from Verduyn, et. al, 1992, Yeast 8, 501-517, see Tables 1, 3, and 4). Seeds are run in 250 mL baffled flasks (50 mL working volume) at 250 rpm and 30.degree. C. The contents of the flasks are harvested at approximately 16-24 hours incubation time with 2.5% v/v inoculum used to start fermenters. Fermenter initial working volume is 1.25 L. The cell dry weight at inoculation is found in Table 5. Fermenter media is outlined in Tables 2, 3, and 4. Carbon substrate (glucose or sucrose) is provided by the addition of 140 g/L at the start of the batch. pH is started at the ambient pH of the media (pH 4-6) and controlled at 3.5 using 28% NH.sub.4OH until 5 mL of ammonium hydroxide solution is added to the 1.25 L batch. At this point, pH control is switched to Ca(OH)2. 1.5 g of calcium hydroxide per 100 mL deionized water is used. Once the 100 mL calcium hydroxide is exhausted pH is allowed to freefall. The fermenter systems are sparged at 0.125 slpm with air targeting 0.125 slpm aeration. Agitation rate is maintained to achieve an oxygen uptake rate of the yeast from 13-22 mmol/L-h. These fermentations are operated such that after the cells achieve a sufficient density, oxygen limitation is achieved and subsequently maintained throughout the rest of the fermentation (e.g., dissolved oxygen less than about 10%).
[0096] Dissolved oxygen is measured using Mettler Toledo INPRO.RTM. 6800 sensor (Mettler-Toledo GmbH, Urdorf, Switzerland), calibrated prior to inoculation. 0% is calibrated by sparging nitrogen, 100% is calibrated using the run conditions in the vessel as detailed above (prior to inoculation).
TABLE-US-00001 TABLE 1 Defined Media for Seed Flask Cultures Chemicals g/L or mL added Ammonium Sulfate 5.0 g/L Magnesium sulfate 0.5 g/L heptahydrate Potassium phosphate 3.0 g/L monobasic (MKP) Glucose 100.0 g/L Trace solution 1.0 mL Vitamin solution 1.0 mL MES buffer (0.1M) 19.0 g/L Glycerol (10% stock) 1.0 mL De-ionized Water 868 g
TABLE-US-00002 TABLE 2 Defined Media for Fermenters Compound Concentration (g/kg) C.sub.6H.sub.12O.sub.6 or Sucrose 140 (NH.sub.4).sub.2SO.sub.4 0.2 KH.sub.2PO.sub.4 0.5 MgSO.sub.4--7H.sub.2O 0.125 Biotin Stock solution (mL) 5 1000.times. Trace Solution (mL) 1
TABLE-US-00003 TABLE 3 Trace Element 1000.times. Stock Solution. Chemical g/1.0 L ZnSO.sub.4.cndot.7H.sub.2O 4.50 MnCl.sub.2.cndot.2H.sub.2O 0.84 CuSO.sub.4.cndot.5H.sub.2O 0.30 FeSO.sub.4.cndot.7H.sub.2O 3.00
TABLE-US-00004 TABLE 4 Vitamin 1000.times. Stock Solution Chemical g/ 1.0 L Biotin (D-) 0.05
[0097] Cell concentration is obtained from an optical density measurement using an established conversion factor between dry cell mass and optical density. Optical density is measured at wavelength of 600 nm with a 1 cm pathlength using a model Genesys20 spectrophotometer (Thermo Scientific). Unless explicitly noted otherwise, an experimentally derived conversion factor of 1.51 OD600 units per 1 g dry cell mass is used to estimate cell dry weight ("CDW").
[0098] Oxygen uptake rate ("OUR") is calculated using methods known to those in the art as described above. For this example, Oxygen and CO2 values are measured by an EGAS L instrument (Sartorious). While a mass spectrometer is not necessarily used, the results obtained by the EGAS L are expected to be substantially the same. Nitrogen value is calculated as 100% less % measured CO2 minus, less % measured Oxygen. Samples are taken at whichever occurred first, 57 h batch time or the reduction of total carbon sources (glucose, fructose and/or sucrose) to <10 g/L (e.g., some batches can be sampled as soon at 33 h if the carbon sources are sufficiently exhausted at this time) and analyzed for biomass growth via OD600, succinate and glucose by high performance liquid chromatography with refractive index and ultraviolet detector.
TABLE-US-00005 TABLE 5 Cell Dry Weight at the beginning and end of fermentation. Initial CDW Final CDW Strain (g/L) (g/L) 1-1 0.1 6 1-2 0.1 5.9 1-3 0.1 5.5 1-4 0.1 6.8 1-5 0.2 5.7 1-8 0.1 5.2
[0099] Table 6 illustrates that a strain with a "ratio of invertase activity to glucose capacity" that is significantly less than 95 produces more succinate than a strain with a "ratio of invertase activity to glucose capacity" that is greater than 95. Accordingly, a strain having a relatively weak promoter of the invertase gene can produce more succinate than a comparable strain having a strong promoter (see also FIG. 3).
TABLE-US-00006 TABLE 6 Glucose consumption, invertase activity, and product formation for selected strains Ratio of invertase activity to glucose capacity (g glucose released/ (g cell dry weight * hour))/(g Invertase glucose Batch Glucose Glucose Succinate Activity consumed/ finish Consumption capacity Succinate Succinate Succinate Specific (g glucose (g cell dry time Rate (g/(g Rate (g/ Titer Yield Rate (g/ released/ weight * Strain (h) (g/(L* h)) CDW * h)) (L * h)) (g/L) (g/g) (g CDW * h)) (g CDW * h)) hour)). 1-1 56.2 2.25 0.51 1.12 60.5 0.497 0.26 No No invertase invertase present present 1-2 56.2 2.15 0.55 1.02 54.9 0.473 0.26 53.97 98.13 1-3 56.2 2.27 0.56 1.07 57.6 0.469 0.26 NM NM 1-4 56.2 2.29 0.54 1.10 57.9 0.480 0.26 9.87 18.28 NM = not measured
[0100] Table 7 illustrates that a strain expressing an invertase gene having the same promoter as strain 1-4 in either a glucose or a sucrose fermentation can achieve a succinate titer equivalent to an equivalent comparable strain without an invertase gene in a glucose fermentation. FIGS. 1 and 2 also show data supporting this conclusion.
TABLE-US-00007 TABLE 7 Succinate titers for glucose and sucrose fermentations of selected strains Sugar Product Sugar Sugar Specific Specific provided Consump- Consump- Rate Batch to tion tion Product Product Product (g/(g finish fermen- Rate Rate (g/(g Rate (g/ Titer Yield CDW * Strain time tation (g/(L*h)) CDW * h)) (L* h)) (g/L) (g/g) h)) 1-5 43.8 Glucose 3.17 0.76 2.44 98.8 0.770 0.58 1-8 44.2 Glucose 3.18 0.77 2.48 101.5 0.779 0.6 1-8 44.2 Sucrose 3.05 0.69 2.43 99.5 0.799 0.55
[0101] The disclosures of each and every patent, patent application, or publication cited herein are hereby incorporated by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and variations.
Sequence CWU
1
1
171993DNAIssatchenkia orientalis 1aacagtatcg atgaaaggtg tacgacttta
taagagggct tttctcgtag ctctttcaaa 60tagtatctca ttgtatacta agatagtttg
tatttgtgtg tgtgtgtcag tgtaagtgtt 120agtatacttg ttttcctctt tcccctagag
ttggtggtgt gttttgttgg aacgtacatt 180agatgcataa tgcgtgacac cgccatgatg
gttgtattct accaatgaga catggccgtt 240gatcctgctg tgtgggtcat gagacatcac
ctcttggggg ggattctcct ataattggca 300ccgtgtatgc ctcaaccact aacttccacc
ctataactga atatattaca taagcaaatc 360tactttttgt ttgtgttgat cgccatcgtt
gaaattcgcg caacttctgg tggctcaacg 420ctgctgttct atcggtatcc taagagatgt
ctttgccctg agtctagggt aaactatcca 480ccttcgttgc tgtttgacta gacagctact
aactttaggg tagtaaatga ataacggctc 540gctctcatga tcacttctct acatcaccct
aacaagtgta ttattttttt ttcaagtggg 600tgttgctgtt ggtgctagcc ttagtgccct
cgttaatagt tgaacaaaca ctggcatttg 660gagtataatg aaaagggatc actacccccc
gcttcctgtt ccgcttctcc cttccggaaa 720aaccacccac cctttctttt cccccactaa
tgtatgaatt tttccgttcc caggggaatg 780gcccacttgg ttctctgtta acccacacaa
ttttgacgca tcccacacac cttttttttt 840tctaccccac actttccctt gaaaaatctc
caatttgaac tggcaatttt caccccccac 900cacttgcatt cattagtgag tcaatccatc
ccgcggtcgg agattcggaa tccacctact 960ggtaatctgt aatctatatt cccgctgacc
ctt 9932690DNAIssatchenkia orientalis
2tagatactgc tcctcctcca atcgaattat tagctgaaac tgttccaact ttgaagagat
60tgggtaaatt aagaccagat tttgaaattt taattgacgg tggtgtcaaa agaggtaccg
120atattttgaa agcagtcgca atcggtggcc aagatgtcag agtttcagtt ggtatgggta
180gacctttctt atatgccaac tcttgctatg gtgaagcagg tgttagaaaa ttaattcaaa
240atctaaagga tgaattagaa atggatatga gattgttggg tgtcactaaa atggaccagc
300tatcttcgaa acatgtcgat actaaacgtt tgattggtag agatgcgatc aactatttgt
360atgataatgt atacagccca atcgaaaccg ttaaattcaa caatgaagat tgattgttgg
420aaatatatta ttcataaagg cgaaaacatt cccttggtat tttattccaa atttatgata
480catagacgta ttttttatat ataaagttat attattaatg attcaagaaa aagttcaaat
540aaactaatgg atcaacctat ttcgaccctt tcttcattgc tacttcttcc ttaagcaaca
600gatgattaag tagatactgt ttttttagcc aatagtatct cgccgaggag ttatacttga
660ctagctcttg ctcaagaatc ttcctaagac
69033435DNAArtificial SequenceFRD variant 3atggctgatg gcaaaacctc
tgcatcagtt gttgctgttg atgctgaacg tgccgctaag 60gaaagagatg cagcagctag
agctatgttg caaggtggtg gtgtctctcc tgctggcaag 120gcacaattgt tgaaaaaggg
tttggttcac actgttccat ataccttaaa ggttgtcgtc 180gcagatccaa aggaaatgga
gaaggcaact gctgacgcag aagaggtttt acaagctgca 240tttcaagtcg tcgacaccct
tttgaacaac tttaacgaaa actcagaagt ttcaagagtc 300aataggttgg cagttggtga
ggaacatcaa atgtctgaaa cattgaaaca cgtcatggcc 360tgttgtcaaa aggtttatca
ttcctccaga ggtgtttttg acccagcagt tggtccatta 420gtccgtgaac ttagagaagc
tgctcacaag ggtaaaactg ttccagccga aagagttaat 480gatttgttat ccaaatgtac
ccttaatgca tctttttcaa ttgatatgtc cagaggtatg 540attgcaagga agcatccaga
cgccatgttg gatttgggtg gtgtcaacaa gggttatggt 600atcgactaca ttgttgaaca
cttaaactct ttgggttatg atgatgtctt tttcgaatgg 660ggtggtgatg ttagagcatc
cggcaaaaac cagttatctc aaccttgggc tgttggtatt 720gttagaccac ctgccttggc
cgacattaga actgttgtcc cagaggacaa aagatccttt 780atccgtgtcg tcagattgaa
caacgaagct attgctacct ctggtgatta tgagaatttg 840gttgaaggtc ctggttctaa
ggtttactct tccaccttca atccaacttc caaaaacttg 900ttggaaccta ccgaagcagg
tatggctcaa gtttctgtca agtgttgctc atgtatctac 960gctgatgctt tagcaacagc
agctttgttg aaaaacgatc ctgctgccgt tagaaggatc 1020ttagataact ggagatatgt
cagagatact gttactgact acaccactta cacaagggaa 1080ggtgaaagag ttgctaagat
gttggaaatt gctaccgaag atgctgaaat gagagcaaag 1140agaatcaagg gctctttacc
agcaagagtt atcattgttg gtggtggttt ggccggttgt 1200tccgcagcta tcgaagcagc
taactgtggc gcccacgtca tcttgttagc aaaggaacca 1260aagttaggtg gtaactctgc
aaaggctacc tccggtatca acgcctgggg tactagagca 1320caagcaaaac aaggtgtcat
ggacggcggc aagtttttcg aaagagatac ccatagatcc 1380ggcaagggtg gtaattgcga
tccatgcctt gttaagactt tgtccgttaa gtcctctgat 1440gcagttaagt ggttatctga
attaggtgtt ccattgactg ttttgtctca attaggtggt 1500gcttcaagga aacgttgtca
ccgtgcacca gataagtctg atggtacacc agtcccagtt 1560ggtttcacca ttatgaaaac
ccttgaaaac cacattgtca acgatttgtc cagacatgtt 1620acagttatga caggtattac
cgtcacagct ttagaatcta catcaagagt cagacctgat 1680ggtgttttag tcaagcatgt
tactggtgtt cacttgattc aggcatctgg tcaatctatg 1740gttttgaatg cagacgctgt
tatcttagct actggtggtt tctccaatga tcatacccca 1800aactcccttt tacaacaata
cgccccacag ttgtcatctt ttccaacaac caatggtgtc 1860tgggcaactg gcgatggtgt
taagatggct tccaagttgg gtgtcgcctt agttgatatg 1920gataaggtcc aattacatcc
taccggcttg ttagacccaa aagatccatc taatagaacc 1980aagtatcttg gtccagaggc
cttaagaggt tccggcggtg tcttgttaaa caaaaacggt 2040gaaagatttg ttaatgaatt
agacttaaga tctgttgtct ctcaagctat catcgcacaa 2100gataatgagt acccaggctc
tggtggttcc aagttcgcat actgtgtttt gaacgaaact 2160gcagcaaagt tattcggcaa
aaacttcctt ggtttctact ggaatagatt aggtcttttc 2220caaaaggttg attccgttgc
tggtttagct aagttgattg gttgtccaga agctaatgtt 2280gttgctacat tgaagcaata
tgaggagtta tcttccaaaa agcttaatcc ttgtccattg 2340actggcaagt ctgtctttcc
ttgtgtttta ggcactcaag gtccatacta tgttgccttg 2400gttaccccat ccattcacta
cactatgggt ggttgtttga tttccccatc tgctgagatg 2460caaaccattg acaactctgg
tgttactcct gtcagacgtc caatcttagg cttattcggt 2520gctggtgaag ttactggcgg
tgtccatggt ggtaacagat taggcggtaa ctctttgtta 2580gaatgtgttg ttttcggcaa
gatcgctggt gacagagctg caaccatctt gcaaaagaaa 2640aacaccggct tatcaatgac
agaatggtct actgtcgtct taagagaagt tagagaaggt 2700ggtgtctatg gtgctggttc
cagagttttg aggtttaaca tgcctggtgc attacagaga 2760actggtttag ctttaggtca
attcatcggt atcagaggtg attgggacgg tcacagattg 2820atcggttact attctccaat
cactttacct gatgatgttg gtgttattgg tatcttagct 2880agagcagaca agggtagatt
ggcagaatgg atttctgcat tgcagccagg tgacgctgtt 2940gagatgaagg cctgcggtgg
tcttatcatt gacagaagat tcgctgaaag acatttcttt 3000ttccgtggtc ataagatcag
aaagttggcc cttatcggtg gtggtactgg tgttgcacca 3060atgttacaaa tcgtcagagc
tgctgtcaaa aagccatttg tcgattcaat tgagtccatt 3120cagttcatct atgctgcaga
ggatgtttcc gagcttacat acagaacctt acttgaatct 3180tacgaagagg aatatggttc
agaaaagttt aagtgtcact tcgttttgaa taacccacca 3240gctcaatgga ctgacggtgt
tggtttcgtt gatactgcat tgttgagatc cgcagttcaa 3300gcaccatcaa atgatttgct
tgttgcaatt tgtggtccac caatcatgca aagagcagtt 3360aagggtgcat tgaaaggttt
aggttacaat atgaatcttg ttagaaccgt tgacgaaact 3420gaaccaccat cataa
343541458DNAIssatchenkia
orientalis 4atgttagctg ctagatcatt aaaggcaaga atgtcaacaa gagctttctc
aactacctca 60attgcaaaaa gaatcgaaaa agatgcattt ggtgacattg aagtcccaaa
tgagaaatat 120tggggtgctc aaactcaaag atctttacaa aatttcaaaa ttggtggtaa
gagagaagtt 180atgccagaac caatcatcaa atcttttggt attttaaaga aggctactgc
taagatcaat 240gctgagtctg gtgctttaga cccaaagtta tctgaagcca tccaacaagc
tgcaaccgaa 300gtttatgaag gtaaactaat ggaccatttc ccattagttg tctttcaaac
cggttctggt 360actcaatcta acatgaatgc caatgaagtc atctctaata gagcaattga
aatcttgggt 420ggtgaattag gctctaaaac tccagtccat cctaatgatc atgttaatat
gtcccaatct 480tctaatgata ctttccctac tgtcatgcat attgcagcag ttacagaagt
ttcatcccat 540ttattaccag aattaactgc actaagagat gcattgcaaa agaaatccga
tgaatttaag 600aatattatca aaatcggtag aacccattta caagatgcaa ctcctttaac
tttaggtcaa 660gaattttctg gttatgttca acaatgtact aatggtatca aaagaatcga
aattgctctt 720gaacatttga gatacttagc tcaaggtggt actgccgttg gtactggtct
taacaccaag 780aaaggttttg ctgaaaaggt tgcaaatgaa gtcactaaat tgactggttt
acaattctat 840accgctccaa ataaattcga agcccttgca gctcacgatg ctgttgttga
aatgtctggt 900gctttgaata ccgttgcagt ctcattattc aaaatcgctc aagatatcag
atatttgggt 960tccggcccaa gatgtggtta tggtgaattg gctttaccag aaaatgaacc
aggttcttcc 1020atcatgccgg gtaaagttaa cccaactcaa aacgaagctt tgactatgct
ttgtacccaa 1080gtctttggta accactcttg tattaccttt gcaggtgctt caggtcaatt
cgaattgaat 1140gtctttaagc cagttatgat ctccaacttg ttatcttcta ttaggttatt
aggtgatggt 1200tgtaattctt ttagaatcca ctgtgttgaa ggtatcattg caaataccga
caagattgat 1260aaattactac atgaatctct catgttagtt actgctttga acccacacat
tggttacgat 1320aaggcttcca agattgcaaa gaatgcacac aagaagggct tgacattgaa
acaatctgca 1380ttggaattag gttacttgac cgaagaacaa ttcaatgaat gggttagacc
agaaaacatg 1440attggtccaa aggattaa
145855797DNAArtificial SequenceURA3-invertase-locus A
transformation sequence 5aaaccataat gcgtgacacc gccatgatgg ttgtattcta
ccaatgagac atggccgctg 60atcctgttgt gtgggtcatg ggacatcacc tcttgggggg
gattctccta taattggcac 120cgtgtatgcc tcaaccacta acttccaccc tataactgaa
tatattacat aagcaaatct 180actttttgtt tgtgttgatc gccatcgttg aaattcgcgc
aacttctggt ggctcaacgc 240tgctgttcta tcggtatcct aagagatgtc tttgccctga
gtctagggta aactatccac 300cttcgttgct gtttgactag acagctacta actttacggt
agtaaatgaa taacggctcg 360ctctcatgat cacttctcta catcacccta acaagtgtat
tatttttttt tcaggtgggt 420gttgctgttg gtgctagcat atggcggccg cggatccctc
gaggagtcca tcggttcctg 480tcagatggga tactcttgac gtggaaaatt caaacagaaa
aaaaacccca ataatgaaaa 540ataacactac gttatatccg tggtatcctc tatcgtatcg
tatcgtagcg tatcgtagcg 600taccgtatca cagtatagtc taatattccg tatcttattg
tatcctatcc tattcgatcc 660tattgtattt cagtgcacca ttttaatttc tattgctata
atgtccttat tagttgccac 720tgtgaggtga ccaatggacg agggcgagcc gttcagaagc
cgcgaagggt gttcttccca 780tgaatttctt aaggagggcg gctcagctcc gagagtgagg
cgagacgtct cggtcagcgt 840atcccccttc ctcggctttt acaaatgatg cgctcttaat
agtgtgtcgt tatccttttg 900gcattgacgg gggagggaaa ttgattgagc gcatccatat
ttttgcggac tgctgaggac 960aatggtggtt tttccgggtg gcgtgggcta caaatgatac
gatggttttt ttcttttcgg 1020agaaggcgta taaaaaggac acggagaacc catttattct
aaaaacagtt gagcttcttt 1080aattattttt tgatataata ttctattatt atatattttc
ttcccaataa aacaaaataa 1140aacaaaacac agcaaaacac aaaaattcta gataaaatgt
taaagttatt gtccttgatg 1200gtcccattag cttctgcagc tgttatccac agacgtgatg
ctaacatttc agctattgca 1260tccgaatgga actccacttc taactcttct tcatctttat
ctttaaacag accagctgtc 1320cattattctc cagaggaagg ttggatgaac gacccaaacg
gtttatggta cgatgctaaa 1380gaggaagatt ggcacatcta ctatcaatac tatcctgatg
cccctcattg gggtttgcca 1440ttgacttggg gtcatgcagt ctccaaagat ttgaccgtct
gggacgaaca aggtgttgca 1500ttcggtccag agtttgaaac agcaggtgcc ttttctggtt
ctatggttat tgattacaat 1560aacacctccg gtttctttaa ctcatccacc gacccaagac
aacgtgtcgt tgccatttgg 1620actttggatt attctggctc tgaaacacaa caattatctt
attctcatga tggtggttat 1680acattcaccg aatattctga caaccctgtc ttagatattg
actcagacgc ttttagagat 1740ccaaaggttt tctggtatca aggtgaagat tccgaatcag
aaggtaactg ggtcatgaca 1800gttgccgaag cagatcgttt ctccgtctta atctactctt
ctccagacct taagaattgg 1860accttagaat caaacttttc cagagaaggc tacttaggct
ataactatga gtgtcctggt 1920ttagttaagg tcccatacgt caaaaacacc acatacgcat
ctgctccagg ctcaaatatc 1980acctcatctg gtccacttca tccaaattct actgtttctt
tctcaaattc atcctctatt 2040gcatggaatg cttcttccgt tccacttaac attactttat
ccaattctac cttggttgat 2100gaaacttctc aattggaaga agttggttac gcatgggtta
tgattgtctc attcaatcct 2160ggctccattt taggcggttc cggtactgaa tacttcatcg
gtgactttaa tggtacacac 2220ttcgagccac ttgataagca aactagattc ttagatttgg
gtaaagatta ctacgctttg 2280caaactttct tcaatacccc aaacgaggtt gacgttttgg
gtatcgcatg ggcctctaat 2340tggcaatatg ctaaccaagt tccaacagat ccatggagat
catccatgtc cttggttaga 2400aacttcacta tcactgaata caacatcaat tctaatacta
ctgcattggt cttgaactct 2460caaccagttt tagattttac ctctttaaga aagaacggca
catcatatac tttagagaat 2520cttacattaa actcctcttc tcacgaggtt ttggaatttg
aagatcctac cggtgttttc 2580gaattttccc ttgaatattc cgtcaacttt accggtattc
acaactgggt ttttaccgac 2640ttgtccttgt atttccaagg tgataaggat tcagatgaat
acttgagact tggttacgaa 2700gctaactcca agcagttctt tttagataga ggtcattcta
acattccatt tgttcaagaa 2760aatccattct tcactcagag actttcagtt tccaatcctc
catcctccaa ctcctccacc 2820ttcgatgtct acggtattgt tgacagaaat atcattgaat
tgtatttcaa caatggtact 2880gttacctcta ctaacacctt tttcttctcc actggtaaca
atattggttc catcattgtt 2940aagtctggtg ttgatgacgt ctatgaaatt gaatcattga
aggttaatca gttttacgtt 3000gactaattaa ttaacatctg aatgtaaaat gaacattaaa
atgaattact aaactttacg 3060tctactttac aatctataaa ctttgtttaa tcatataacg
aaatacacta atacacaatc 3120ctgtacgtat gtaatacttt tatccatcaa ggattgagaa
aaaaaagtaa tgattccctg 3180ggccattaaa acttagaccc ccaagcttgg ataggtcact
ctctattttc gtttctccct 3240tccctgatag aagggtgata tgtaattaag aataatatat
aattttataa taaaagaatt 3300catagcctca tgaaatcagc catttgcttt tgttcaacga
tcttttgaaa ttgttgttgt 3360tcttggtagt taagttgatc catcttggct tatgttgtgt
gtatgttgta gttattctta 3420gtatattcct gtcctgagtt tagtgaaaca taatatcgcc
ttgaaatgaa aatgctgaaa 3480ttcgtcgaca tacaattttt caaacttttt ttttttcttg
gtgcacggac atgtttttaa 3540aggaagtact ctataccagt tattcttcac aaatttaatt
gctggagaat agatcttcaa 3600cgctttaata aagtagtttg tttgtcaagg atggcgtcat
acaaagaaag atcagaatca 3660cacacttccc ctgttgctag gagacttttc tccatcatgg
aggaaaagaa gtctaacctt 3720tgtgcatcat tggatattac tgaaactgaa aagcttctct
ctattttgga cactattggt 3780ccttacatct gtctagttaa aacacacatc gatattgttt
ctgattttac gtatgaagga 3840actgtgttgc ctttgaagga gcttgccaag aaacataatt
ttatgatttt tgaagataga 3900aaatttgctg atattggtaa cactgttaaa aatcaatata
aatctggtgt cttccgtatt 3960gccgaatggg ctgacatcac taatgcacat ggtgtaacgg
gtgcaggtat tgtttctggc 4020ttgaaggagg cagcccaaga aacaaccagt gaacctagag
gtttgctaat gcttgctgag 4080ttatcatcaa agggttcttt agcatatggt gaatatacag
aaaaaacagt agaaattgct 4140aaatctgata aagagtttgt cattggtttt attgcgcaac
acgatatggg cggtagagaa 4200gaaggttttg actggatcat tatgactcca ggggttggtt
tagatgacaa aggtgatgca 4260cttggtcaac aatatagaac tgttgatgaa gttgtaaaga
ctggaacgga tatcataatt 4320gttggtagag gtttgtacgg tcaaggaaga gatcctatag
agcaagctaa aagataccaa 4380caagctggtt ggaatgctta tttaaacaga tttaaatgat
tcttacacaa agatttgata 4440catgtacact agtttaaata agcatgaaaa gaattacaca
agcaaaaaaa aaaaaataaa 4500tgaggtactt tacgttcacc tacaaccaaa aaaactagat
agagtaaaat cttaagattt 4560agaaaaagtt gtttaacaaa ggctttagta tgtgaatttt
taatgtagca aagcgataac 4620taataaacat aaacaaaagt atggttttct ttatcagtca
aatcattatc gattgattgt 4680tccgcgtatc tgcagatagc ctcatgaaat cagccatttg
cttttgttca acgatctttt 4740gaaattgttg ttgttcttgg tagttaagtt gatccatctt
ggcttatgtt gtgtgtatgt 4800tgtagttatt cttagtatat tcctgtcctg agtttagtga
aacataatat cgccttgaaa 4860tgaaaatgct gaaattcgtc gacatacaat ttttcaaact
tttttttttt cttggtgcac 4920ggacatgttt ttaaaggaag tactctatac cagttattct
tcacaaattt aattgctgga 4980gaatagatct tcaacgcccc gggggatctg gatccgcggc
cgcgagctct aatgattcaa 5040gaaaaagttc aaataaacta atggatcaac ctatttcgac
cctttcttca ttgctacttc 5100ttccttaagc aacagatgat taagtagata ctgttttttt
agccaatagt atctcgccga 5160ggagttatac ttgactagct cttgctcaag aatcttccta
agacgtacta gcctagcata 5220gtaatctgtt tgtttctgta ttgtttgttc taactgttct
acagtcattg aatcaatatc 5280tccaatgtct tcgacgttga caactttccc ccccttggca
gcattctctt ttttgttgga 5340atacgacatt aaagattcct tgattttctg ggtaccttca
atgaccattg agggattaaa 5400tttgatttct ttgattttat aatggtcggc tattagctct
tccacttcgt catcatgatc 5460atcagatatg tcacgttgcc ttttcaattt attaaaattg
tttatcagtt tattgtgatc 5520ttgtatcaat tcattgcgta ctcttttctc aatatcaaaa
gctattttct tcccgctaga 5580ctcaaaatca actctgaagt cattttctcg ctggaattca
tgtatttcat ggattaattc 5640tctattgata ttctcgtatg catcctgtaa actgttgccg
ttgatattat gaaccgcctt 5700taaatgtttc aataaggcat ctgctctagt aaatgccttc
agacattcag gtaataaaca 5760gtaaaatggc ttctcggctg tatgcgtcct aatgttt
57976609PRTKluyveromyces lactis 6Met Leu Lys Leu
Leu Ser Leu Met Val Pro Leu Ala Ser Ala Ala Val1 5
10 15Ile His Arg Arg Asp Ala Asn Ile Ser Ala
Ile Ala Ser Glu Trp Asn 20 25
30Ser Thr Ser Asn Ser Ser Ser Ser Leu Ser Leu Asn Arg Pro Ala Val
35 40 45His Tyr Ser Pro Glu Glu Gly Trp
Met Asn Asp Pro Asn Gly Leu Trp 50 55
60Tyr Asp Ala Lys Glu Glu Asp Trp His Ile Tyr Tyr Gln Tyr Tyr Pro65
70 75 80Asp Ala Pro His Trp
Gly Leu Pro Leu Thr Trp Gly His Ala Val Ser 85
90 95Lys Asp Leu Thr Val Trp Asp Glu Gln Gly Val
Ala Phe Gly Pro Glu 100 105
110Phe Glu Thr Ala Gly Ala Phe Ser Gly Ser Met Val Ile Asp Tyr Asn
115 120 125Asn Thr Ser Gly Phe Phe Asn
Ser Ser Thr Asp Pro Arg Gln Arg Val 130 135
140Val Ala Ile Trp Thr Leu Asp Tyr Ser Gly Ser Glu Thr Gln Gln
Leu145 150 155 160Ser Tyr
Ser His Asp Gly Gly Tyr Thr Phe Thr Glu Tyr Ser Asp Asn
165 170 175Pro Val Leu Asp Ile Asp Ser
Asp Ala Phe Arg Asp Pro Lys Val Phe 180 185
190Trp Tyr Gln Gly Glu Asp Ser Glu Ser Glu Gly Asn Trp Val
Met Thr 195 200 205Val Ala Glu Ala
Asp Arg Phe Ser Val Leu Ile Tyr Ser Ser Pro Asp 210
215 220Leu Lys Asn Trp Thr Leu Glu Ser Asn Phe Ser Arg
Glu Gly Tyr Leu225 230 235
240Gly Tyr Asn Tyr Glu Cys Pro Gly Leu Val Lys Val Pro Tyr Val Lys
245 250 255Asn Thr Thr Tyr Ala
Ser Ala Pro Gly Ser Asn Ile Thr Ser Ser Gly 260
265 270Pro Leu His Pro Asn Ser Thr Val Ser Phe Ser Asn
Ser Ser Ser Ile 275 280 285Ala Trp
Asn Ala Ser Ser Val Pro Leu Asn Ile Thr Leu Ser Asn Ser 290
295 300Thr Leu Val Asp Glu Thr Ser Gln Leu Glu Glu
Val Gly Tyr Ala Trp305 310 315
320Val Met Ile Val Ser Phe Asn Pro Gly Ser Ile Leu Gly Gly Ser Gly
325 330 335Thr Glu Tyr Phe
Ile Gly Asp Phe Asn Gly Thr His Phe Glu Pro Leu 340
345 350Asp Lys Gln Thr Arg Phe Leu Asp Leu Gly Lys
Asp Tyr Tyr Ala Leu 355 360 365Gln
Thr Phe Phe Asn Thr Pro Asn Glu Val Asp Val Leu Gly Ile Ala 370
375 380Trp Ala Ser Asn Trp Gln Tyr Ala Asn Gln
Val Pro Thr Asp Pro Trp385 390 395
400Arg Ser Ser Met Ser Leu Val Arg Asn Phe Thr Ile Thr Glu Tyr
Asn 405 410 415Ile Asn Ser
Asn Thr Thr Ala Leu Val Leu Asn Ser Gln Pro Val Leu 420
425 430Asp Phe Thr Ser Leu Arg Lys Asn Gly Thr
Ser Tyr Thr Leu Glu Asn 435 440
445Leu Thr Leu Asn Ser Ser Ser His Glu Val Leu Glu Phe Glu Asp Pro 450
455 460Thr Gly Val Phe Glu Phe Ser Leu
Glu Tyr Ser Val Asn Phe Thr Gly465 470
475 480Ile His Asn Trp Val Phe Thr Asp Leu Ser Leu Tyr
Phe Gln Gly Asp 485 490
495Lys Asp Ser Asp Glu Tyr Leu Arg Leu Gly Tyr Glu Ala Asn Ser Lys
500 505 510Gln Phe Phe Leu Asp Arg
Gly His Ser Asn Ile Pro Phe Val Gln Glu 515 520
525Asn Pro Phe Phe Thr Gln Arg Leu Ser Val Ser Asn Pro Pro
Ser Ser 530 535 540Asn Ser Ser Thr Phe
Asp Val Tyr Gly Ile Val Asp Arg Asn Ile Ile545 550
555 560Glu Leu Tyr Phe Asn Asn Gly Thr Val Thr
Ser Thr Asn Thr Phe Phe 565 570
575Phe Ser Thr Gly Asn Asn Ile Gly Ser Ile Ile Val Lys Ser Gly Val
580 585 590Asp Asp Val Tyr Glu
Ile Glu Ser Leu Lys Val Asn Gln Phe Tyr Val 595
600 605Asp7703DNAIssatchenkia orientalis 7gagtccatcg
gttcctgtca gatgggatac tcttgacgtg gaaaattcaa acagaaaaaa 60aaccccaata
atgaaaaata acactacgtt atatccgtgg tatcctctat cgtatcgtat 120cgtagcgtat
cgtagcgtac cgtatcacag tatagtctaa tattccgtat cttattgtat 180cctatcctat
tcgatcctat tgtatttcag tgcaccattt taatttctat tgctataatg 240tccttattag
ttgccactgt gaggtgacca atggacgagg gcgagccgtt cagaagccgc 300gaagggtgtt
cttcccatga atttcttaag gagggcggct cagctccgag agtgaggcga 360gacgtctcgg
ttagcgtatc ccccttcctc ggcttttaca aatgatgcgc tcttaatagt 420gtgtcgttat
ccttttggca ttgacggggg agggaaattg attgagcgca tccatatttt 480ggcggactgc
tgaggacaat ggtggttttt ccgggtggcg tgggctacaa atgatacgat 540ggtttttttc
ttttcggaga aggcgtataa aaaggacacg gagaacccat ttattctaat 600aacagttgag
cttctttaat tatttgttaa tataatattc tattattata tattttcttc 660ccaataaaac
aaaataaaac aaaacacagc aaaacacaaa aat
70385598DNAArtificial SequenceURA3-invertase-locus A transformation
sequence 8aaaccataat gcgtgacacc gccatgatgg ttgtattcta ccaatgagac
atggccgctg 60atcctgttgt gtgggtcatg ggacatcacc tcttgggggg gattctccta
taattggcac 120cgtgtatgcc tcaaccacta acttccaccc tataactgaa tatattacat
aagcaaatct 180actttttgtt tgtgttgatc gccatcgttg aaattcgcgc aacttctggt
ggctcaacgc 240tgctgttcta tcggtatcct aagagatgtc tttgccctga gtctagggta
aactatccac 300cttcgttgct gtttgactag acagctacta actttacggt agtaaatgaa
taacggctcg 360ctctcatgat cacttctcta catcacccta acaagtgtat tatttttttt
tcaggtgggt 420gttgctgttg gtgctagcat atggcggccg cggatccctc gaggtggtca
atgtttaact 480agaattatag gccaatctta tcttctccga attcattgca gccaccatca
atttaacatt 540ggttttcaaa taatcagtta aacctgacag tgattaccag ccacggggat
tcgagaaatt 600cgagcaattc gaaagagccg caaaaaaata agcaagaaaa aagaaaataa
aaaacaaaaa 660aaacatgaaa aaaacatgaa aaaaacatga aaaaaaaaaa aaagaaagaa
aaaaaaaatc 720aattttttca tcagaggcat tggtaaaaca ctctgtgctg tcgcgacacg
acgaacggga 780tgccatggta cttgtttcgg gtgtctctcc tcctttggaa actctttccg
ctctcgaaaa 840aattaatacc gtagaggagg ggacatgaat agaatcgtat ataacagggt
cattccctga 900tgctgtcttt gtaaggagcc atttattgtc atttgtttat accaacccca
ttgaacaaac 960aagaaaatct agataaaatg ttaaagttat tgtccttgat ggtcccatta
gcttctgcag 1020ctgttatcca cagacgtgat gctaacattt cagctattgc atccgaatgg
aactccactt 1080ctaactcttc ttcatcttta tctttaaaca gaccagctgt ccattattct
ccagaggaag 1140gttggatgaa cgacccaaac ggtttatggt acgatgctaa agaggaagat
tggcacatct 1200actatcaata ctatcctgat gcccctcatt ggggtttgcc attgacttgg
ggtcatgcag 1260tctccaaaga tttgaccgtc tgggacgaac aaggtgttgc attcggtcca
gagtttgaaa 1320cagcaggtgc cttttctggt tctatggtta ttgattacaa taacacctcc
ggtttcttta 1380actcatccac cgacccaaga caacgtgtcg ttgccatttg gactttggat
tattctggct 1440ctgaaacaca acaattatct tattctcatg atggtggtta tacattcacc
gaatattctg 1500acaaccctgt cttagatatt gactcagacg cttttagaga tccaaaggtt
ttctggtatc 1560aaggtgaaga ttccgaatca gaaggtaact gggtcatgac agttgccgaa
gcagatcgtt 1620tctccgtctt aatctactct tctccagacc ttaagaattg gaccttagaa
tcaaactttt 1680ccagagaagg ctacttaggc tataactatg agtgtcctgg tttagttaag
gtcccatacg 1740tcaaaaacac cacatacgca tctgctccag gctcaaatat cacctcatct
ggtccacttc 1800atccaaattc tactgtttct ttctcaaatt catcctctat tgcatggaat
gcttcttccg 1860ttccacttaa cattacttta tccaattcta ccttggttga tgaaacttct
caattggaag 1920aagttggtta cgcatgggtt atgattgtct cattcaatcc tggctccatt
ttaggcggtt 1980ccggtactga atacttcatc ggtgacttta atggtacaca cttcgagcca
cttgataagc 2040aaactagatt cttagatttg ggtaaagatt actacgcttt gcaaactttc
ttcaataccc 2100caaacgaggt tgacgttttg ggtatcgcat gggcctctaa ttggcaatat
gctaaccaag 2160ttccaacaga tccatggaga tcatccatgt ccttggttag aaacttcact
atcactgaat 2220acaacatcaa ttctaatact actgcattgg tcttgaactc tcaaccagtt
ttagatttta 2280cctctttaag aaagaacggc acatcatata ctttagagaa tcttacatta
aactcctctt 2340ctcacgaggt tttggaattt gaagatccta ccggtgtttt cgaattttcc
cttgaatatt 2400ccgtcaactt taccggtatt cacaactggg tttttaccga cttgtccttg
tatttccaag 2460gtgataagga ttcagatgaa tacttgagac ttggttacga agctaactcc
aagcagttct 2520ttttagatag aggtcattct aacattccat ttgttcaaga aaatccattc
ttcactcaga 2580gactttcagt ttccaatcct ccatcctcca actcctccac cttcgatgtc
tacggtattg 2640ttgacagaaa tatcattgaa ttgtatttca acaatggtac tgttacctct
actaacacct 2700ttttcttctc cactggtaac aatattggtt ccatcattgt taagtctggt
gttgatgacg 2760tctatgaaat tgaatcattg aaggttaatc agttttacgt tgactaatta
attaacatct 2820gaatgtaaaa tgaacattaa aatgaattac taaactttac gtctacttta
caatctataa 2880actttgttta atcatataac gaaatacact aatacacaat cctgtacgta
tgtaatactt 2940ttatccatca aggattgaga aaaaaaagta atgattccct gggccattaa
aacttagacc 3000cccaagcttg gataggtcac tctctatttt cgtttctccc ttccctgata
gaagggtgat 3060atgtaattaa gaataatata taattttata ataaaagaat tcatagcctc
atgaaatcag 3120ccatttgctt ttgttcaacg atcttttgaa attgttgttg ttcttggtag
ttaagttgat 3180ccatcttggc ttatgttgtg tgtatgttgt agttattctt agtatattcc
tgtcctgagt 3240ttagtgaaac ataatatcgc cttgaaatga aaatgctgaa attcgtcgac
atacaatttt 3300tcaaactttt tttttttctt ggtgcacgga catgttttta aaggaagtac
tctataccag 3360ttattcttca caaatttaat tgctggagaa tagatcttca acgctttaat
aaagtagttt 3420gtttgtcaag gatggcgtca tacaaagaaa gatcagaatc acacacttcc
cctgttgcta 3480ggagactttt ctccatcatg gaggaaaaga agtctaacct ttgtgcatca
ttggatatta 3540ctgaaactga aaagcttctc tctattttgg acactattgg tccttacatc
tgtctagtta 3600aaacacacat cgatattgtt tctgatttta cgtatgaagg aactgtgttg
cctttgaagg 3660agcttgccaa gaaacataat tttatgattt ttgaagatag aaaatttgct
gatattggta 3720acactgttaa aaatcaatat aaatctggtg tcttccgtat tgccgaatgg
gctgacatca 3780ctaatgcaca tggtgtaacg ggtgcaggta ttgtttctgg cttgaaggag
gcagcccaag 3840aaacaaccag tgaacctaga ggtttgctaa tgcttgctga gttatcatca
aagggttctt 3900tagcatatgg tgaatataca gaaaaaacag tagaaattgc taaatctgat
aaagagtttg 3960tcattggttt tattgcgcaa cacgatatgg gcggtagaga agaaggtttt
gactggatca 4020ttatgactcc aggggttggt ttagatgaca aaggtgatgc acttggtcaa
caatatagaa 4080ctgttgatga agttgtaaag actggaacgg atatcataat tgttggtaga
ggtttgtacg 4140gtcaaggaag agatcctata gagcaagcta aaagatacca acaagctggt
tggaatgctt 4200atttaaacag atttaaatga ttcttacaca aagatttgat acatgtacac
tagtttaaat 4260aagcatgaaa agaattacac aagcaaaaaa aaaaaaataa atgaggtact
ttacgttcac 4320ctacaaccaa aaaaactaga tagagtaaaa tcttaagatt tagaaaaagt
tgtttaacaa 4380aggctttagt atgtgaattt ttaatgtagc aaagcgataa ctaataaaca
taaacaaaag 4440tatggttttc tttatcagtc aaatcattat cgattgattg ttccgcgtat
ctgcagatag 4500cctcatgaaa tcagccattt gcttttgttc aacgatcttt tgaaattgtt
gttgttcttg 4560gtagttaagt tgatccatct tggcttatgt tgtgtgtatg ttgtagttat
tcttagtata 4620ttcctgtcct gagtttagtg aaacataata tcgccttgaa atgaaaatgc
tgaaattcgt 4680cgacatacaa tttttcaaac tttttttttt tcttggtgca cggacatgtt
tttaaaggaa 4740gtactctata ccagttattc ttcacaaatt taattgctgg agaatagatc
ttcaacgccc 4800cgggggatct ggatccgcgg ccgcgagctc taatgattca agaaaaagtt
caaataaact 4860aatggatcaa cctatttcga ccctttcttc attgctactt cttccttaag
caacagatga 4920ttaagtagat actgtttttt tagccaatag tatctcgccg aggagttata
cttgactagc 4980tcttgctcaa gaatcttcct aagacgtact agcctagcat agtaatctgt
ttgtttctgt 5040attgtttgtt ctaactgttc tacagtcatt gaatcaatat ctccaatgtc
ttcgacgttg 5100acaactttcc cccccttggc agcattctct tttttgttgg aatacgacat
taaagattcc 5160ttgattttct gggtaccttc aatgaccatt gagggattaa atttgatttc
tttgatttta 5220taatggtcgg ctattagctc ttccacttcg tcatcatgat catcagatat
gtcacgttgc 5280cttttcaatt tattaaaatt gtttatcagt ttattgtgat cttgtatcaa
ttcattgcgt 5340actcttttct caatatcaaa agctattttc ttcccgctag actcaaaatc
aactctgaag 5400tcattttctc gctggaattc atgtatttca tggattaatt ctctattgat
attctcgtat 5460gcatcctgta aactgttgcc gttgatatta tgaaccgcct ttaaatgttt
caataaggca 5520tctgctctag taaatgcctt cagacattca ggtaataaac agtaaaatgg
cttctcggct 5580gtatgcgtcc taatgttt
55989453DNAIssatchenkia orientalis 9attgcagcca ccatcaattt
aacattggtt ttcaaataat cagttaaacc tgacagtgat 60taccagccac ggggattcga
gaaattcgag caattcgaaa gagccgcaaa aaaataagca 120agaaaaaaga aaataaaaaa
caaaaaaaac atgaaaaaaa catgaaaaaa acatgaaaaa 180aaaaaaaaag aaagaaaaaa
aaaatcaatt ttttcatcag aggcattggt aaaacactct 240gtgctgtcgc gacacgacga
acgggatgcc atggtacttg tttcgggtgt ctctcctcct 300ttggaaactc tttccgctct
cgaaaaaatt aataccgtag aggaggggac atgaatagaa 360tcgtatataa cagggtcatt
ccctgatgct gtctttgtaa ggagccattt attgtcattt 420gtttatacca accccattga
acaaacaaga aaa 453106015DNAArtificial
SequenceURA3-invertase-locus A transformation sequence 10aaaccataat
gcgtgacacc gccatgatgg ttgtattcta ccaatgagac atggccgctg 60atcctgttgt
gtgggtcatg ggacatcacc tcttgggggg gattctccta taattggcac 120cgtgtatgcc
tcaaccacta acttccaccc tataactgaa tatattacat aagcaaatct 180actttttgtt
tgtgttgatc gccatcgttg aaattcgcgc aacttctggt ggctcaacgc 240tgctgttcta
tcggtatcct aagagatgtc tttgccctga gtctagggta aactatccac 300cttcgttgct
gtttgactag acagctacta actttacggt agtaaatgaa taacggctcg 360ctctcatgat
cacttctcta catcacccta acaagtgtat tatttttttt tcaggtgggt 420gttgctgttg
gtgctagcat atggcggccg cggatccctc gagtttggat gaggcattca 480atgtctccat
gtttgaagac cagagaaagt gtaggtaaca caatagccat ggacaattag 540cgaatccgaa
tatgtgtact aacattgtgc ctccaactga cagctggtcg aataatgatt 600gaaatcatca
aacatatcag agatatatcc cagaaccatt taacaactgt tattgtatgt 660atgcattact
gttttacatt gtgtctttcc aaatactaac attgccatta tccaatccat 720acatatatat
atatatagat tacacaacgc aaacctcgac agtcgattgg tttacatcgt 780gtaagcagcc
ttttacggaa tatgatggac aaccagatcc tcatagatgg agacaatgtg 840cagatagatt
ccatttatga tgacattgat gtgtttttaa atgcaaaata atctcctata 900taagtgagtt
ccgagtctcc ccatgcttat cttatcgtgt tttttctgta aattaggtca 960aagtattcaa
acaattccct caatctttcc cattttttgc tgagccccca tatcatcaaa 1020ttaacgtatc
aaatttccta attagtagcc ccgtatgtta aattagacat gtgtgactgc 1080catcccggac
agcctaccca atgacacccg cgcaccgcac atattgtgtt tagtgcgcgc 1140cgtctgctga
agcgactccc tgtttgggag gaaccgaggg cgggttgccc gatcccttgc 1200ccctcgctcc
tcctcctggg ctcccccttg cagagggaca ccgaggggat ccctcgtgtg 1260agagcttgga
ggtggatggt ggtcaatttt ctcatttgat tgaaagattt gttatattga 1320aaattcagtt
tgtggaagtt gtgattaaaa ggttttactg tttgttctgt agacacattc 1380aatatctaga
taaaatgtta aagttattgt ccttgatggt cccattagct tctgcagctg 1440ttatccacag
acgtgatgct aacatttcag ctattgcatc cgaatggaac tccacttcta 1500actcttcttc
atctttatct ttaaacagac cagctgtcca ttattctcca gaggaaggtt 1560ggatgaacga
cccaaacggt ttatggtacg atgctaaaga ggaagattgg cacatctact 1620atcaatacta
tcctgatgcc cctcattggg gtttgccatt gacttggggt catgcagtct 1680ccaaagattt
gaccgtctgg gacgaacaag gtgttgcatt cggtccagag tttgaaacag 1740caggtgcctt
ttctggttct atggttattg attacaataa cacctccggt ttctttaact 1800catccaccga
cccaagacaa cgtgtcgttg ccatttggac tttggattat tctggctctg 1860aaacacaaca
attatcttat tctcatgatg gtggttatac attcaccgaa tattctgaca 1920accctgtctt
agatattgac tcagacgctt ttagagatcc aaaggttttc tggtatcaag 1980gtgaagattc
cgaatcagaa ggtaactggg tcatgacagt tgccgaagca gatcgtttct 2040ccgtcttaat
ctactcttct ccagacctta agaattggac cttagaatca aacttttcca 2100gagaaggcta
cttaggctat aactatgagt gtcctggttt agttaaggtc ccatacgtca 2160aaaacaccac
atacgcatct gctccaggct caaatatcac ctcatctggt ccacttcatc 2220caaattctac
tgtttctttc tcaaattcat cctctattgc atggaatgct tcttccgttc 2280cacttaacat
tactttatcc aattctacct tggttgatga aacttctcaa ttggaagaag 2340ttggttacgc
atgggttatg attgtctcat tcaatcctgg ctccatttta ggcggttccg 2400gtactgaata
cttcatcggt gactttaatg gtacacactt cgagccactt gataagcaaa 2460ctagattctt
agatttgggt aaagattact acgctttgca aactttcttc aataccccaa 2520acgaggttga
cgttttgggt atcgcatggg cctctaattg gcaatatgct aaccaagttc 2580caacagatcc
atggagatca tccatgtcct tggttagaaa cttcactatc actgaataca 2640acatcaattc
taatactact gcattggtct tgaactctca accagtttta gattttacct 2700ctttaagaaa
gaacggcaca tcatatactt tagagaatct tacattaaac tcctcttctc 2760acgaggtttt
ggaatttgaa gatcctaccg gtgttttcga attttccctt gaatattccg 2820tcaactttac
cggtattcac aactgggttt ttaccgactt gtccttgtat ttccaaggtg 2880ataaggattc
agatgaatac ttgagacttg gttacgaagc taactccaag cagttctttt 2940tagatagagg
tcattctaac attccatttg ttcaagaaaa tccattcttc actcagagac 3000tttcagtttc
caatcctcca tcctccaact cctccacctt cgatgtctac ggtattgttg 3060acagaaatat
cattgaattg tatttcaaca atggtactgt tacctctact aacacctttt 3120tcttctccac
tggtaacaat attggttcca tcattgttaa gtctggtgtt gatgacgtct 3180atgaaattga
atcattgaag gttaatcagt tttacgttga ctaattaatt aacatctgaa 3240tgtaaaatga
acattaaaat gaattactaa actttacgtc tactttacaa tctataaact 3300ttgtttaatc
atataacgaa atacactaat acacaatcct gtacgtatgt aatactttta 3360tccatcaagg
attgagaaaa aaaagtaatg attccctggg ccattaaaac ttagaccccc 3420aagcttggat
aggtcactct ctattttcgt ttctcccttc cctgatagaa gggtgatatg 3480taattaagaa
taatatataa ttttataata aaagaattca tagcctcatg aaatcagcca 3540tttgcttttg
ttcaacgatc ttttgaaatt gttgttgttc ttggtagtta agttgatcca 3600tcttggctta
tgttgtgtgt atgttgtagt tattcttagt atattcctgt cctgagttta 3660gtgaaacata
atatcgcctt gaaatgaaaa tgctgaaatt cgtcgacata caatttttca 3720aacttttttt
ttttcttggt gcacggacat gtttttaaag gaagtactct ataccagtta 3780ttcttcacaa
atttaattgc tggagaatag atcttcaacg ctttaataaa gtagtttgtt 3840tgtcaaggat
ggcgtcatac aaagaaagat cagaatcaca cacttcccct gttgctagga 3900gacttttctc
catcatggag gaaaagaagt ctaacctttg tgcatcattg gatattactg 3960aaactgaaaa
gcttctctct attttggaca ctattggtcc ttacatctgt ctagttaaaa 4020cacacatcga
tattgtttct gattttacgt atgaaggaac tgtgttgcct ttgaaggagc 4080ttgccaagaa
acataatttt atgatttttg aagatagaaa atttgctgat attggtaaca 4140ctgttaaaaa
tcaatataaa tctggtgtct tccgtattgc cgaatgggct gacatcacta 4200atgcacatgg
tgtaacgggt gcaggtattg tttctggctt gaaggaggca gcccaagaaa 4260caaccagtga
acctagaggt ttgctaatgc ttgctgagtt atcatcaaag ggttctttag 4320catatggtga
atatacagaa aaaacagtag aaattgctaa atctgataaa gagtttgtca 4380ttggttttat
tgcgcaacac gatatgggcg gtagagaaga aggttttgac tggatcatta 4440tgactccagg
ggttggttta gatgacaaag gtgatgcact tggtcaacaa tatagaactg 4500ttgatgaagt
tgtaaagact ggaacggata tcataattgt tggtagaggt ttgtacggtc 4560aaggaagaga
tcctatagag caagctaaaa gataccaaca agctggttgg aatgcttatt 4620taaacagatt
taaatgattc ttacacaaag atttgataca tgtacactag tttaaataag 4680catgaaaaga
attacacaag caaaaaaaaa aaaataaatg aggtacttta cgttcaccta 4740caaccaaaaa
aactagatag agtaaaatct taagatttag aaaaagttgt ttaacaaagg 4800ctttagtatg
tgaattttta atgtagcaaa gcgataacta ataaacataa acaaaagtat 4860ggttttcttt
atcagtcaaa tcattatcga ttgattgttc cgcgtatctg cagatagcct 4920catgaaatca
gccatttgct tttgttcaac gatcttttga aattgttgtt gttcttggta 4980gttaagttga
tccatcttgg cttatgttgt gtgtatgttg tagttattct tagtatattc 5040ctgtcctgag
tttagtgaaa cataatatcg ccttgaaatg aaaatgctga aattcgtcga 5100catacaattt
ttcaaacttt ttttttttct tggtgcacgg acatgttttt aaaggaagta 5160ctctatacca
gttattcttc acaaatttaa ttgctggaga atagatcttc aacgccccgg 5220gggatctgga
tccgcggccg cgagctctaa tgattcaaga aaaagttcaa ataaactaat 5280ggatcaacct
atttcgaccc tttcttcatt gctacttctt ccttaagcaa cagatgatta 5340agtagatact
gtttttttag ccaatagtat ctcgccgagg agttatactt gactagctct 5400tgctcaagaa
tcttcctaag acgtactagc ctagcatagt aatctgtttg tttctgtatt 5460gtttgttcta
actgttctac agtcattgaa tcaatatctc caatgtcttc gacgttgaca 5520actttccccc
ccttggcagc attctctttt ttgttggaat acgacattaa agattccttg 5580attttctggg
taccttcaat gaccattgag ggattaaatt tgatttcttt gattttataa 5640tggtcggcta
ttagctcttc cacttcgtca tcatgatcat cagatatgtc acgttgcctt 5700ttcaatttat
taaaattgtt tatcagttta ttgtgatctt gtatcaattc attgcgtact 5760cttttctcaa
tatcaaaagc tattttcttc ccgctagact caaaatcaac tctgaagtca 5820ttttctcgct
ggaattcatg tatttcatgg attaattctc tattgatatt ctcgtatgca 5880tcctgtaaac
tgttgccgtt gatattatga accgccttta aatgtttcaa taaggcatct 5940gctctagtaa
atgccttcag acattcaggt aataaacagt aaaatggctt ctcggctgta 6000tgcgtcctaa
tgttt
601511921DNAIssatchenkia orientalis 11tttggatgag gcattcaatg tctccatgtt
tgaagaccag agaaagtgta ggtaacacaa 60tagccatgga caattagcga atccgaatat
gtgtactaac attgtgcctc caactgacag 120ctggtcgaat aatgattgaa atcatcaaac
atatcagaga tatatcccag aaccatttaa 180caactgttat tgtatgtatg cattactgtt
ttacattgtg tctttccaaa tactaacatt 240gccattatcc aatccataca tatatatata
tatagattac acaacgcaaa cctcgacagt 300cgattggttt acatcgtgta agcagccttt
tacggaatat gatggacaac cagatcctca 360tagatggaga caatgtgcag atagattcca
tttatgatga cattgatgtg tttttaaatg 420caaaataatc tcctatataa gtgagttccg
agtctcccca tgcttatctt atcgtgtttt 480ttctgtaaat taggtcaaag tattcaaaca
attccctcaa tctttcccat tttttgctga 540gcccccatat catcaaatta acgtatcaaa
tttcctaatt agtagccccg tatgttaaat 600tagacatgtg tgactgccat cccggacagc
ctacccaatg acacccgcgc accgcacata 660ttgtgtttag tgcgcgccgt ctgctgaagc
gactccctgt ttgggaggaa ccgagggcgg 720gttgcccgat cccttgcccc tcgctcctcc
tcctgggctc ccccttgcag agggacaccg 780aggggatccc tcgtgtgaga gcttggaggt
ggatggtggt caattttctc atttgattga 840aagatttgtt atattgaaaa ttcagtttgt
ggaagttgtg attaaaaggt tttactgttt 900gttctgtaga cacattcaat a
921129630DNAArtificial
Sequenceinvertase-CRE recombinase transformation
plasmidmisc_feature(3086)..(3086)n is a, c, g, or
tmisc_feature(5897)..(5897)n is a, c, g, or tmisc_feature(6283)..(6283)n
is a, c, g, or tmisc_feature(6314)..(6314)n is a, c, g, or
tmisc_feature(6343)..(6343)n is a, c, g, or tmisc_feature(6347)..(6347)n
is a, c, g, or tmisc_feature(6371)..(6371)n is a, c, g, or
tmisc_feature(6525)..(6525)n is a, c, g, or tmisc_feature(6543)..(6543)n
is a, c, g, or tmisc_feature(7386)..(7386)n is a, c, g, or
tmisc_feature(7397)..(7397)n is a, c, g, or t 12ctgacgcgcc ctgtagcggc
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 60ccgctacact tgccagcgcc
ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 120ccacgttcgc cggctttccc
cgtcaagctc taaatcgggg gctcccttta gggttccgat 180ttagtgcttt acggcacctc
gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 240ggccatcgcc ctgatagacg
gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 300gtggactctt gttccaaact
ggaacaacac tcaaccctat ctcggtctat tcttttgatt 360tataagggat tttgccgatt
tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 420ttaacgcgaa ttttaacaaa
atattaacgc ttacaatttc cattcgccat tcaggctgcg 480caactgttgg gaagggcgat
cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540gggatgtgct gcaaggcgat
taagttgggt aacgccaggg ttttcccagt cacgacgttg 600taaaacgacg gccagtgagc
gcgcgtaata cgactcacta tagggcgaat tggtcgagga 660gtccatcggt tcctgtcaga
tgggatactc ttgacgtgga aaattcaaac agaaaaaaaa 720ccccaataat gaaaaataac
actacgttat atccgtggta tcctctatcg tatcgtatcg 780tagcgtatcg tagcgtaccg
tatcacagta tagtctaata ttccgtatct tattgtatcc 840tatcctattc gatcctattg
tatttcagtg caccatttta atttctattg ctataatgtc 900cttattagtt gccactgtga
ggtgaccaat ggacgagggc gagccgttca gaagccgcga 960agggtgttct tcccatgaat
ttcttaagga gggcggctca gctccgagag tgaggcgaga 1020cgtctcggtc agcgtatccc
ccttcctcgg cttttacaaa tgatgcgctc ttaatagtgt 1080gtcgttatcc ttttggcatt
gacgggggag ggaaattgat tgagcgcatc catatttttg 1140cggactgctg aggacaatgg
tggtttttcc gggtggcgtg ggctacaaat gatacgatgg 1200tttttttctt ttcggagaag
gcgtataaaa aggacacgga gaacccattt attctaaaaa 1260cagttgagct tctttaatta
ttttttgata taatattcta ttattatata ttttcttccc 1320aataaaacaa aataaaacaa
aacacagcaa aacacaaaaa ggatccatgt ctaatttact 1380tactgttcac caaaacttgc
ctgcattacc agttgacgca acctccgatg aagtcagaaa 1440gaaccttatg gatatgttta
gagatagaca agctttctcc gaacatactt ggaaaatgtt 1500attatccgtt tgtagatcct
gggccgcttg gtgtaaactt aacaatagaa aatggtttcc 1560tgctgaacca gaagacgtca
gagattactt actttactta caagctagag gtttggctgt 1620taaaactatc caacaacact
taggtcaatt gaatatgtta cacagaagat ccggtttacc 1680aagaccatcc gattccaacg
cagtttccct tgttatgaga agaattagaa aagaaaatgt 1740tgacgctggt gaaagagcta
aacaagcatt agcatttgaa agaaccgatt tcgatcaagt 1800tagatcctta atggaaaatt
ccgatagatg tcaagatatt agaaacttag ctttcttagg 1860tattgcttac aacacattat
taagaatcgc tgaaattgct agaattagag ttaaagatat 1920ttcaagaacc gatggcggta
gaatgttaat ccacattggc agaacaaaaa ccttagtctc 1980cacagcaggc gtcgaaaaag
cattatcatt aggtgttact aaattagttg aacgttggat 2040ttccgtttcc ggtgttgcag
atgacccaaa caactactta ttctgtcgtg ttagaaaaaa 2100tggtgttgcc gctccttccg
ctacctcaca attatccaca agagcattag aaggcatttt 2160tgaagctacc cacagactta
tttatggtgc aaaagacgat tccggtcaaa gatatttagc 2220ttggtctggt cattccgcta
gagttggtgc cgcaagagac atggcaagag ctggtgtttc 2280tattcctgaa attatgcaag
ccggtggttg gactaatgtt aacattgtta tgaactatat 2340cagaaactta gattccgaaa
caggtgctat ggttagatta cttgaagacg gtgattaagt 2400taattaacat ctgaatgtaa
aatgaacatt aaaatgaatt actaaacttt acgtctactt 2460tacaatctat aaactttgtt
taatcatata acgaaataca ctaatacaca atcctgtacg 2520tatgtaatac ttttatccat
caaggattga gaaaaaaaag taatgattcc ctgggccatt 2580aaaacttaga cccccaagct
tggataggtc actctctatt ttcgtttctc ccttccctga 2640tagaagggtg atatgtaatt
aagaataata tataatttta taataaaaga attcggcaga 2700tctggatcga tcccccgggc
tgcatgcaac ggcaacatca atgtccacgt ttacacacct 2760acatttatat ctatatttat
atttatattt atttatttat gctacttagc ttctatagtt 2820agttaatgca ctcacgatat
tcaaaattga cacccttcaa ctactcccta ctattgtcta 2880ctactgtcta ctactcctct
ttactatagc tgctcccaat aggctccacc aataggctct 2940gtcaatacat tttgcgccgc
cacctttcag gttgtgtcac tcctgaagga ccatattggg 3000taatcgtgca atttctggaa
gagagtgccg cgagaagtga ggcccccact gtaaatcctc 3060gagggggcat ggagtatggg
gcatgnagga tggaggatgg gggggggggg ggaaaatagg 3120tagcgaaagg acccgctatc
accccacccg gagaactcgt tgccgggaag tcatatttcg 3180acactccggg gagtctataa
aaggcgggtt ttgtcttttg ccagttgatg ttgctgagag 3240gacttgtttg ccgtttcttc
cgatttaaca gtatagaatc aaccactgtt aattatacac 3300gttatactaa cacaacaaaa
acaaaaacaa cgacaacaac aacaacctgc aggaaatgct 3360tttgcaagct ttccttttcc
ttttggctgg ttttgcagcc aaaatatctg catcaatgac 3420aaacgaaact agcgatagac
ctttggtcca cttcacaccc aacaagggct ggatgaatga 3480cccaaatggg ttgtggtacg
atgaaaaaga tgccaaatgg catctgtact ttcaatacaa 3540cccaaatgac accgtatggg
gtacgccatt gttttggggc catgctactt ccgatgattt 3600gactcattgg gaagatgaac
ccattgctat cgctcccaag cgtaacgatt caggtgcttt 3660ctctggctcc atggtggttg
attacaacaa cactagtggg tttttcaatg atactattga 3720tccaagacaa agatgcgttg
caatttggac ttataacact cctgaaagtg aagagcaata 3780cattagctat tcccttgatg
gtggttacac ttttactgaa taccaaaaga accctgtttt 3840agctgccaac tccactcaat
tcagagatcc aaaggtgttc tggtatgaac cttctcaaaa 3900atggattatg acggctgcca
aatcacaaga ctacaaaatt gaaatttact cctcggatga 3960cttgaagtcc tggaagttag
aatctgcatt tgccaatgaa ggtttcttag gctaccaata 4020tgaatgtcca ggtttgattg
aagtcccaac tgagcaagat ccttccaaat cctattgggt 4080catgtttatt tctatcaacc
caggtgcacc tgctggcggt tccttcaacc aatattttgt 4140tggatccttc aatggtactc
attttgaagc gtttgacaat caatctagag tggtagattt 4200tggtaaggac tactatgcct
tgcaaacttt cttcaacact gacccaacct acggttcagc 4260attaggtatt gcctgggctt
caaactggga gtacagtgcc tttgtcccaa ctaacccatg 4320gagatcatcc atgtctttgg
tccgcaagtt ttcgttgaac actgaatatc aagctaatcc 4380agagactgaa ttgatcaatt
tgaaagccga accaatattg aacattagta atgctggccc 4440ctggtctcgt tttgctacta
acacaactct aactaaggcc aattcttaca atgtcgattt 4500gagcaactcg actggtaccc
tagagtttga gttggtttac gctgttaaca ccacacaaac 4560catatccaaa tccgtctttg
ccgacttatc actttggttc aagggtttag aagatcctga 4620agaatatttg agaatgggtt
ttgaagccag tgcttcttcc ttctttttgg accgtggtaa 4680ctctaaggtc aagtttgtca
aggagaaccc atatttcaca aacagaatgt ctgtcaacaa 4740ccaaccattc aagtctgaga
acgacctaag ttactataaa gtgtacggcc tactggatca 4800aaacatcttg gaattgtact
tcaacgatgg agatgtggtt tctacaaata cctacttcat 4860gaccaccggc aacgctctag
gatctgtgaa catgaccact ggtgtcgata atttgttcta 4920cattgacaag ttccaagtaa
gggaagtaaa atagcctgca ggcacgtccg acggcggccc 4980acgggtccca ggcctcggag
atccgtcccc cttttccttt gtcgatatca tgtaattagt 5040tatgtcacgc ttacattcac
gccctccccc cacatccgct ctaaccgaaa aggaaggagt 5100tagacaacct gaagtctagg
tccctattta tttttttata gttatgttag tattaagaac 5160gttatttata tttcaaattt
ttcttttttt tctgtacaga cgcgtgtacg catgtaacat 5220tatactgaaa accttgcttg
agaaggtttt gggacgctcg aaggctttaa tttgcaagct 5280gaattcccgg gccttaccgt
cgacgaattt cagcattttc atttcaaggc gatattatgt 5340ttcactaaac tcaggacagg
aatatactaa gaataactac aacatacaca caacataagc 5400caagatggat caacttaact
accaagaaca acaacaattt caaaagatcg ttgaacaaaa 5460gcaaatggct gatttcatga
ggctatgaat tcgcccttga tctgggtgta tactgcacaa 5520cctcattgtt cgggaatttg
attctcatct cacatacagg cctgtagtat tgcgccctct 5580ccttctcctt ctccttctcc
ttctccaaga gagacttctc tctcatcgcc ctcgtcatca 5640atggctgctc gctgtattgt
cgttggagca tctcccgata cttctgcaac tgtgataaac 5700tcatctcagg tgacccatcc
gattctgtat cggtgtctcc atctggggct acatctcggg 5760ccagtctaga tttaaacttt
gcagaacctt cactttgggg gatatacact agtgtctctc 5820ccgtgactac atcaccgaca
ccctcaactg taccattatt attgtcattg ttttcctcta 5880agttctcgct ttggtcntca
tccatctctc cttcgggtgc tgtatcactc ttgatgattt 5940ctctaaccct aatacggaga
ctgtgattgc ctgaaataat acccacatct ttcaacttct 6000gatgaagtga atctccagag
atgaccttca tcagcacttg cacatcaacc acatcaccct 6060ccttttgagc atccctcatg
attccataga ctacatcccg tagcgtctcc ttgttcttgt 6120acttcttaac aacagtctcg
ccacagacat ggcccctgat aatcacctcc tgtctctcct 6180catggccatc ctggtcgcca
ttgtcttcgt cgctcggctc aattgccaat gtagcaccct 6240gtggaagatt gcttagtctg
tatggaacag actcatcaac tcntttgcca ttatgcatta 6300acttgtactt tcgnccttgg
ctaagttgaa aatgtttaca tcnwtcntca agtacattgg 6360acatgattgt ncctgcattg
acatttgtcc ggtaagtcct aaacccactc gctagattca 6420ctgtaggcat attcaatcac
gttccgtttg aaaaaaagga aaccaattta ttatctccag 6480aaatagttgg cgtcttgcat
cttgtttggt cttgatcttt cgtgnttttt tttttttctg 6540tcnttttttt tctcctctct
ccaacttttt gatttttagt gtaccaaatc gcactgctta 6600tccacattca tcataaagrg
ggggggagaa gaggggcaga aaataaaagg ccatgtcacg 6660tgcctgtgca tttatttgtg
tgtgtgtcac gtgctcaaaa tgtctttttt ttacgttttt 6720aacattttcc ctttctgtag
ttgaatccat ttgcatgagt cgtacatrat gtttgctgta 6780tttacgttaa gacactaatt
caaatgacaa acagctatta ttcttagcca ttaatgcatt 6840tttgcaaatc tttaactgga
tttaactatg gctaggtraa tttgttctgg acatcattgc 6900cttgacttgt tttagtgccg
atgtccttat cacttacact cgtaacacaa cacaacagca 6960gctaatgttg ttgtgtatcg
cttgaccctt aataactgat tcttttttga tgaatgttaa 7020gaagaaacaa acaaraaaat
aaaatcaaaa caggcttctt ttgacctctt tcaagagaag 7080gttttcttgg ttgtttcata
taccaagatc tgaatatctt ctattattat acaaaccact 7140gattatacaa atctattcat
cgacagtatg arctacgaaa acacactgat aaaragagtc 7200atttcttccc cttctttttc
tttttctttt tcttcttctt cttagtatcc ccatcttcat 7260taactccacc aagtagatcc
tctacacccc ccatggccgt taaaaaatgt tcacgaaaga 7320aatccatatc attattctta
ccatccatta aactgtttag atagatggtg atcatctccc 7380ttgcantgtc tatatcntca
acgtcgagta aatgcgacgc aatggtaccc agcttttgtt 7440ccctttagtg agggttaatt
gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt 7500gaaattgtta tccgctcaca
attccacaca acatacgagc cggaagcata aagtgtaaag 7560cctggggtgc ctaatgagtg
agctaactca cattaattgc gttgcgctca ctgcccgctt 7620tccagtcggg aaacctgtcg
tgccagctgc attaatgaat cggccaacgc gcggggagag 7680gcggtttgcg tattgggcgc
tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 7740ttcggctgcg gcgagcggta
tcagctcact caaaggcggt aatacggtta tccacagaat 7800caggggataa cgcaggaaag
aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 7860aaaaggccgc gttgctggcg
tttttccata ggctccgccc ccctgacgag catcacaaaa 7920atcgacgctc aagtcagagg
tggcgaaacc cgacaggact ataaagatac caggcgtttc 7980cccctggaag ctccctcgtg
cgctctcctg ttccgaccct gccgcttacc ggatacctgt 8040ccgcctttct cccttcggga
agcgtggcgc tttctcatag ctcacgctgt aggtatctca 8100gttcggtgta ggtcgttcgc
tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 8160accgctgcgc cttatccggt
aactatcgtc ttgagtccaa cccggtaaga cacgacttat 8220cgccactggc agcagccact
ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 8280cagagttctt gaagtggtgg
cctaactacg gctacactag aaggacagta tttggtatct 8340gcgctctgct gaagccagtt
accttcggaa aaagagttgg tagctcttga tccggcaaac 8400aaaccaccgc tggtagcggt
ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 8460aaggatctca agaagatcct
ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 8520actcacgtta agggattttg
gtcatgagat tatcaaaaag gatcttcacc tagatccttt 8580taaattaaaa atgaagtttt
aaatcaatct aaagtatata tgagtaaact tggtctgaca 8640gttaccaatg cttaatcagt
gaggcaccta tctcagcgat ctgtctattt cgttcatcca 8700tagttgcctg actccccgtc
gtgtagataa ctacgatacg ggagggctta ccatctggcc 8760ccagtgctgc aatgataccg
cgagacccac gctcaccggc tccagattta tcagcaataa 8820accagccagc cggaagggcc
gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 8880agtctattaa ttgttgccgg
gaagctagag taagtagttc gccagttaat agtttgcgca 8940acgttgttgc cattgctaca
ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 9000tcagctccgg ttcccaacga
tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 9060cggttagctc cttcggtcct
ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 9120tcatggttat ggcagcactg
cataattctc ttactgtcat gccatccgta agatgctttt 9180ctgtgactgg tgagtactca
accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 9240gctcttgccc ggcgtcaata
cgggataata ccgcgccaca tagcagaact ttaaaagtgc 9300tcatcattgg aaaacgttct
tcggggcgaa aactctcaag gatcttaccg ctgttgagat 9360ccagttcgat gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca 9420gcgtttctgg gtgagcaaaa
acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 9480cacggaaatg ttgaatactc
atactcttcc tttttcaata ttattgaagc atttatcagg 9540gttattgtct catgagcgga
tacatatttg aatgtattta gaaaaataaa caaatagggg 9600ttccgcgcac atttccccga
aaagtgccac 9630136760DNAArtificial
Sequencemelbiase-invertase-locus A transformation
sequencemisc_feature(3991)..(3991)n is a, c, g, or t 13atcgctagcc
ttagtgccct cgttaatagt tgaacaaaca ctggcatttg gagtataatg 60aaaagggatc
actacccccc gcttcctgtt ccgcttctcc cttccggaaa aaccacccac 120cctttctttt
cccccactaa tgtatgaatt tttccgttcc caggggaatg gcccacttgg 180ttctctgtta
acccacacaa ttttgacgca tcccacacac cttttttttt tctaccccac 240actttccctt
gaaaaatctc caatttgaac tggcaatttt caccccccac cacttgcatt 300cattagtgag
tcaatccatc ccgcggtcgg agattcggaa tccacctact ggtaatctgt 360aatctatatt
cccgctgacc ctttataaat gaactattgt cgtcaattgc ggtagtgctc 420caacaaattg
taaggacctt ctttaacctt ttcgattcaa tccatctcca cataaaccta 480gttgcacaca
gctagcatat ggcggccgcg gatccctcga gtttggatga ggcattcaat 540gtctccatgt
ttgaagacca gagaaagtgt aggtaacaca atagccatgg acaattagcg 600aatccgaata
tgtgtactaa cattgtgcct ccaactgaca gctggtcgaa taatgattga 660aatcatcaaa
catatcagag atatatccca gaaccattta acaactgtta ttgtatgtat 720gcattactgt
tttacattgt gtctttccaa atactaacat tgccattatc caatccatac 780atatatatat
atatagatta cacaacgcaa acctcgacag tcgattggtt tacatcgtgt 840aagcagcctt
ttacggaata tgatggacaa ccagatcctc atagatggag acaatgtgca 900gatagattcc
atttatgatg acattgatgt gtttttaaat gcaaaataat ctcctatata 960agtgagttcc
gagtctcccc atgcttatct tatcgtgttt tttctgtaaa ttaggtcaaa 1020gtattcaaac
aattccctca atctttccca ttttttgctg agcccccata tcatcaaatt 1080aacgtatcaa
atttcctaat tagtagcccc gtatgttaaa ttagacatgt gtgactgcca 1140tcccggacag
cctacccaat gacacccgcg caccgcacat attgtgttta gtgcgcgccg 1200tctgctgaag
cgactccctg tttgggagga accgagggcg ggttgcccga tcccttgccc 1260ctcgctcctc
ctcctgggct cccccttgca gagggacacc gaggggatcc ctcgtgtgag 1320agcttggagg
tggatggtgg tcaattttct catttgattg aaagatttgt tatattgaaa 1380attcagtttg
tggaagttgt gattaaaagg ttttactgtt tgttctgtag acacattcaa 1440tatctagata
aaatgttaaa gttattgtcc ttgatggtcc cattagcttc tgcagctgtt 1500atccacagac
gtgatgctaa catttcagct attgcatccg aatggaactc cacttctaac 1560tcttcttcat
ctttatcttt aaacagacca gctgtccatt attctccaga ggaaggttgg 1620atgaacgacc
caaacggttt atggtacgat gctaaagagg aagattggca catctactat 1680caatactatc
ctgatgcccc tcattggggt ttgccattga cttggggtca tgcagtctcc 1740aaagatttga
ccgtctggga cgaacaaggt gttgcattcg gtccagagtt tgaaacagca 1800ggtgcctttt
ctggttctat ggttattgat tacaataaca cctccggttt ctttaactca 1860tccaccgacc
caagacaacg tgtcgttgcc atttggactt tggattattc tggctctgaa 1920acacaacaat
tatcttattc tcatgatggt ggttatacat tcaccgaata ttctgacaac 1980cctgtcttag
atattgactc agacgctttt agagatccaa aggttttctg gtatcaaggt 2040gaagattccg
aatcagaagg taactgggtc atgacagttg ccgaagcaga tcgtttctcc 2100gtcttaatct
actcttctcc agaccttaag aattggacct tagaatcaaa cttttccaga 2160gaaggctact
taggctataa ctatgagtgt cctggtttag ttaaggtccc atacgtcaaa 2220aacaccacat
acgcatctgc tccaggctca aatatcacct catctggtcc acttcatcca 2280aattctactg
tttctttctc aaattcatcc tctattgcat ggaatgcttc ttccgttcca 2340cttaacatta
ctttatccaa ttctaccttg gttgatgaaa cttctcaatt ggaagaagtt 2400ggttacgcat
gggttatgat tgtctcattc aatcctggct ccattttagg cggttccggt 2460actgaatact
tcatcggtga ctttaatggt acacacttcg agccacttga taagcaaact 2520agattcttag
atttgggtaa agattactac gctttgcaaa ctttcttcaa taccccaaac 2580gaggttgacg
ttttgggtat cgcatgggcc tctaattggc aatatgctaa ccaagttcca 2640acagatccat
ggagatcatc catgtccttg gttagaaact tcactatcac tgaatacaac 2700atcaattcta
atactactgc attggtcttg aactctcaac cagttttaga ttttacctct 2760ttaagaaaga
acggcacatc atatacttta gagaatctta cattaaactc ctcttctcac 2820gaggttttgg
aatttgaaga tcctaccggt gttttcgaat tttcccttga atattccgtc 2880aactttaccg
gtattcacaa ctgggttttt accgacttgt ccttgtattt ccaaggtgat 2940aaggattcag
atgaatactt gagacttggt tacgaagcta actccaagca gttcttttta 3000gatagaggtc
attctaacat tccatttgtt caagaaaatc cattcttcac tcagagactt 3060tcagtttcca
atcctccatc ctccaactcc tccaccttcg atgtctacgg tattgttgac 3120agaaatatca
ttgaattgta tttcaacaat ggtactgtta cctctactaa cacctttttc 3180ttctccactg
gtaacaatat tggttccatc attgttaagt ctggtgttga tgacgtctat 3240gaaattgaat
cattgaaggt taatcagttt tacgttgact aattaattaa catctgaatg 3300taaaatgaac
attaaaatga attactaaac tttacgtcta ctttacaatc tataaacttt 3360gtttaatcat
ataacgaaat acactaatac acaatcctgt acgtatgtaa tacttttatc 3420catcaaggat
tgagaaaaaa aagtaatgat tccctgggcc attaaaactt agacccccaa 3480gcttggatag
gtcactctct attttcgttt ctcccttccc tgatagaagg gtgatatgta 3540attaagaata
atatataatt ttataataaa agaattcgcc cttacctgca gggataactt 3600cgtataatgt
atgctatacg aagttatgct gcaacggcaa catcaatgtc cacgtttaca 3660cacctacatt
tatatctata tttatattta tatttattta tttatgctac ttagcttcta 3720tagttagtta
atgcactcac gatattcaaa attgacaccc ttcaactact ccctactatt 3780gtctactact
gtctactact cctctttact atagctgctc ccaataggct ccaccaatag 3840gctctgtcaa
tacattttgc gccgccacct ttcaggttgt gtcactcctg aaggaccata 3900ttgggtaatc
gtgcaatttc tggaagagag tgccgcgaga agtgaggccc ccactgtaaa 3960tcctcgaggg
ggcatggagt atggggcatg naggatggag gatggggggg gggggggaaa 4020ataggtagcg
aaaggacccg ctatcacccc acccggagaa ctcgttgccg ggaagtcata 4080tttcgacact
ccggggagtc tataaaaggc gggttttgtc ttttgccagt tgatgttgct 4140gagaggactt
gtttgccgtt tcttccgatt taacagtata gaatcaacca ctgttaatta 4200tacacgttat
actaacacaa caaaaacaaa aacaacgaca acaacaacaa caatgtttgc 4260tttctacttt
ctcaccgcat gcaccacttt gaagggtgtt ttcggagttt ctccgagtta 4320caatggtctt
ggtctcaccc cacagatggg ttgggacagc tggaatacgt ttgcctgcga 4380tgtcagtgaa
cagctacttc tagacactgc tgatagaatt tctgacttgg ggctaaagga 4440tatgggttac
aagtatgtca tcctagatga ctgttggtct agcggcaggg attccgacgg 4500tttcctcgtt
gcagacaagc acaaatttcc caacggtatg ggccatgttg cagaccacct 4560gcataataac
agctttcttt tcggtatgta ttcgtctgct ggtgagtaca cctgtgctgg 4620gtaccctggg
tctctggggc gtgaggaaga agatgctcaa ttctttgcaa ataaccgcgt 4680tgactacttg
aagtatgata attgttacaa taaaggtcaa tttggtacac cagacgtttc 4740ttaccaccgt
tacaaggcca tgtcagatgc tttgaataaa actggtaggc ctattttcta 4800ttctctatgt
aactggggtc aggatttgac attttactgg ggctctggta tcgccaattc 4860ttggagaatg
agcggagata ttactgctga gttcacccgt ccagatagca gatgtccctg 4920tgacggtgac
gaatatgatt gcaagtacgc cggtttccat tgttctatta tgaatattct 4980taacaaggca
gctccaatgg ggcaaaatgc aggtgttggt ggttggaacg atctggacaa 5040tctagaggtc
ggagtcggta atttgactga cgatgaggaa aaggcccatt tctctatgtg 5100ggcaatggta
aagtccccac ttatcattgg tgccgacgtg aatcacttaa aggcatcttc 5160gtactcgatc
tacagtcaag cctctgtcat cgcaattaat caagatccaa agggtattcc 5220agccacaaga
gtctggagat attatgtttc agacaccgat gaatatggac aaggtgaaat 5280tcaaatgtgg
agtggtccgc ttgacaatgg tgaccaagtg gttgctttat tgaatggagg 5340aagcgtagca
agaccaatga acacgacctt ggaagagatt ttctttgaca gcaatttggg 5400ttcaaaggaa
ctgacatcga cttgggatat ttacgactta tgggccaaca gagttgacaa 5460ctctacggcg
tctgctatcc ttgaacagaa taaggcagcc accggtattc tctacaatgc 5520tacagagcag
tcttataaag acggtttgtc taagaatgat acaagactgt ttggccagaa 5580aattggtagt
ctttctccaa atgctatact taacacaact gttccagctc atggtatcgc 5640cttctatagg
ttgagaccct cggcttaagc tcaatgttga gcaaagcagg acgagaaaaa 5700aaaaaataat
gattgttaag aagttcatga aaaaaaaaag gaaaaatact caaatactta 5760taacagagtg
attaaataat aaacggcagt ataccctatc aggtattgag atagttttat 5820ttttgtaggt
atataatctg aagcctttga actattttct cgtatatatc atggagtata 5880cattgcatta
gcaacattgc atactagttc ataacttcgt ataatgtatg ctatacgaag 5940ttattaatta
acaagggcga attccttgat ttatatacac ctttgcgagc tctaatgatt 6000caagaaaaag
ttcaaataaa ctaatggatc aacctatttc gaccctttct tcattgctac 6060ttcttcctta
agcaacagat gattaagtag atactgtttt tttagccaat agtatctcgc 6120cgaggagtta
tacttgacta gctcttgctc aagaatcttc ctaagacgta ctagcctagc 6180atagtaatct
gtttgtttct gtattgtttg ttctaactgt tctacagtca ttgaatcaat 6240atctccaatg
tcttcgacgt tgacaacttt cccccccttg gcagcattct cttttttgtt 6300ggaatacgac
attaaagatt ccttgatttt ctgggtacct tcaatgacca ttgagggatt 6360aaatttgatt
tctttgattt tataatggtc ggctattagc tcttccactt cgtcatcatg 6420atcatcagat
atgtcacgtt gccttttcaa tttattaaaa ttgtttatca gtttattgtg 6480atcttgtatc
aattcattgc gtactctttt ctcaatatca aaagctattt tcttcccgct 6540agactcaaaa
tcaactctga agtcattttc tcgctggaat tcatgtattt catggattaa 6600ttctctattg
atattctcgt atgcatcctg taaactgttg ccgttgatat tatgaaccgc 6660ctttaaatgt
ttcaataagg catctgctct agtaaatgcc ttcagacatt caggtaataa 6720acagtaaaat
ggcttctcgg ctgtatgcgt cctaatgttt
676014575PRTIssatchenkia orientalis 14Met Thr Asp Lys Ile Ser Leu Gly Thr
Tyr Leu Phe Glu Lys Leu Lys1 5 10
15Glu Ala Gly Ser Tyr Ser Ile Phe Gly Val Pro Gly Asp Phe Asn
Leu 20 25 30Ala Leu Leu Asp
His Val Lys Glu Val Glu Gly Ile Arg Trp Val Gly 35
40 45Asn Ala Asn Glu Leu Asn Ala Gly Tyr Glu Ala Asp
Gly Tyr Ala Arg 50 55 60Ile Asn Gly
Phe Ala Ser Leu Ile Thr Thr Phe Gly Val Gly Glu Leu65 70
75 80Ser Ala Val Asn Ala Ile Ala Gly
Ser Tyr Ala Glu His Val Pro Leu 85 90
95Ile His Ile Val Gly Met Pro Ser Leu Ser Ala Met Lys Asn
Asn Leu 100 105 110Leu Leu His
His Thr Leu Gly Asp Thr Arg Phe Asp Asn Phe Thr Glu 115
120 125Met Ser Lys Lys Ile Ser Ala Lys Val Glu Ile
Val Tyr Asp Leu Glu 130 135 140Ser Ala
Pro Lys Leu Ile Asn Asn Leu Ile Glu Thr Ala Tyr His Thr145
150 155 160Lys Arg Pro Val Tyr Leu Gly
Leu Pro Ser Asn Phe Ala Asp Glu Leu 165
170 175Val Pro Ala Ala Leu Val Lys Glu Asn Lys Leu His
Leu Glu Glu Pro 180 185 190Leu
Asn Asn Pro Val Ala Glu Glu Glu Phe Ile His Asn Val Val Glu 195
200 205Met Val Lys Lys Ala Glu Lys Pro Ile
Ile Leu Val Asp Ala Cys Ala 210 215
220Ala Arg His Asn Ile Ser Lys Glu Val Arg Glu Leu Ala Lys Leu Thr225
230 235 240Lys Phe Pro Val
Phe Thr Thr Pro Met Gly Lys Ser Thr Val Asp Glu 245
250 255Asp Asp Glu Glu Phe Phe Gly Leu Tyr Leu
Gly Ser Leu Ser Ala Pro 260 265
270Asp Val Lys Asp Ile Val Gly Pro Thr Asp Cys Ile Leu Ser Leu Gly
275 280 285Gly Leu Pro Ser Asp Phe Asn
Thr Gly Ser Phe Ser Tyr Gly Tyr Thr 290 295
300Thr Lys Asn Val Val Glu Phe His Ser Asn Tyr Cys Lys Phe Lys
Ser305 310 315 320Ala Thr
Tyr Glu Asn Leu Met Met Lys Gly Ala Val Gln Arg Leu Ile
325 330 335Ser Glu Leu Lys Asn Ile Lys
Tyr Ser Asn Val Ser Thr Leu Ser Pro 340 345
350Pro Lys Ser Lys Phe Ala Tyr Glu Ser Ala Lys Val Ala Pro
Glu Gly 355 360 365Ile Ile Thr Gln
Asp Tyr Leu Trp Lys Arg Leu Ser Tyr Phe Leu Lys 370
375 380Pro Arg Asp Ile Ile Val Thr Glu Thr Gly Thr Ser
Ser Phe Gly Val385 390 395
400Leu Ala Thr His Leu Pro Arg Asp Ser Lys Ser Ile Ser Gln Val Leu
405 410 415Trp Gly Ser Ile Gly
Phe Ser Leu Pro Ala Ala Val Gly Ala Ala Phe 420
425 430Ala Ala Glu Asp Ala His Lys Gln Thr Gly Glu Gln
Glu Arg Arg Thr 435 440 445Val Leu
Phe Ile Gly Asp Gly Ser Leu Gln Leu Thr Val Gln Ser Ile 450
455 460Ser Asp Ala Ala Arg Trp Asn Ile Lys Pro Tyr
Ile Phe Ile Leu Asn465 470 475
480Asn Arg Gly Tyr Thr Ile Glu Lys Leu Ile His Gly Arg His Glu Asp
485 490 495Tyr Asn Gln Ile
Gln Pro Trp Asp His Gln Leu Leu Leu Lys Leu Phe 500
505 510Ala Asp Lys Thr Gln Tyr Glu Asn His Val Val
Lys Ser Ala Lys Asp 515 520 525Leu
Asp Ala Leu Met Lys Asp Glu Ala Phe Asn Lys Glu Asp Lys Ile 530
535 540Arg Val Ile Glu Leu Phe Leu Asp Glu Phe
Asp Ala Pro Glu Ile Leu545 550 555
560Val Ala Gln Ala Lys Leu Ser Asp Glu Ile Asn Ser Lys Ala Ala
565 570
57515532PRTSaccharomyces cerevisiae 15Met Leu Leu Gln Ala Phe Leu Phe Leu
Leu Ala Gly Phe Ala Ala Lys1 5 10
15Ile Ser Ala Ser Met Thr Asn Glu Thr Ser Asp Arg Pro Leu Val
His 20 25 30Phe Thr Pro Asn
Lys Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr 35
40 45Asp Glu Lys Asp Ala Lys Trp His Leu Tyr Phe Gln
Tyr Asn Pro Asn 50 55 60Asp Thr Val
Trp Gly Thr Pro Leu Phe Trp Gly His Ala Thr Ser Asp65 70
75 80Asp Leu Thr Asn Trp Glu Asp Gln
Pro Ile Ala Ile Ala Pro Lys Arg 85 90
95Asn Asp Ser Gly Ala Phe Ser Gly Ser Met Val Val Asp Tyr
Asn Asn 100 105 110Thr Ser Gly
Phe Phe Asn Asp Thr Ile Asp Pro Arg Gln Arg Cys Val 115
120 125Ala Ile Trp Thr Tyr Asn Thr Pro Glu Ser Glu
Glu Gln Tyr Ile Ser 130 135 140Tyr Ser
Leu Asp Gly Gly Tyr Thr Phe Thr Glu Tyr Gln Lys Asn Pro145
150 155 160Val Leu Ala Ala Asn Ser Thr
Gln Phe Arg Asp Pro Lys Val Phe Trp 165
170 175Tyr Glu Pro Ser Gln Lys Trp Ile Met Thr Ala Ala
Lys Ser Gln Asp 180 185 190Tyr
Lys Ile Glu Ile Tyr Ser Ser Asp Asp Leu Lys Ser Trp Lys Leu 195
200 205Glu Ser Ala Phe Ala Asn Glu Gly Phe
Leu Gly Tyr Gln Tyr Glu Cys 210 215
220Pro Gly Leu Ile Glu Val Pro Thr Glu Gln Asp Pro Ser Lys Ser Tyr225
230 235 240Trp Val Met Phe
Ile Ser Ile Asn Pro Gly Ala Pro Ala Gly Gly Ser 245
250 255Phe Asn Gln Tyr Phe Val Gly Ser Phe Asn
Gly Thr His Phe Glu Ala 260 265
270Phe Asp Asn Gln Ser Arg Val Val Asp Phe Gly Lys Asp Tyr Tyr Ala
275 280 285Leu Gln Thr Phe Phe Asn Thr
Asp Pro Thr Tyr Gly Ser Ala Leu Gly 290 295
300Ile Ala Trp Ala Ser Asn Trp Glu Tyr Ser Ala Phe Val Pro Thr
Asn305 310 315 320Pro Trp
Arg Ser Ser Met Ser Leu Val Arg Lys Phe Ser Leu Asn Thr
325 330 335Glu Tyr Gln Ala Asn Pro Glu
Thr Glu Leu Ile Asn Leu Lys Ala Glu 340 345
350Pro Ile Leu Asn Ile Ser Asn Ala Gly Pro Trp Ser Arg Phe
Ala Thr 355 360 365Asn Thr Thr Leu
Thr Lys Ala Asn Ser Tyr Asn Val Asp Leu Ser Asn 370
375 380Ser Thr Gly Thr Leu Glu Phe Glu Leu Val Tyr Ala
Val Asn Thr Thr385 390 395
400Gln Thr Ile Ser Lys Ser Val Phe Ala Asp Leu Ser Leu Trp Phe Lys
405 410 415Gly Leu Glu Asp Pro
Glu Glu Tyr Leu Arg Met Gly Phe Glu Val Ser 420
425 430Ala Ser Ser Phe Phe Leu Asp Arg Gly Asn Ser Lys
Val Lys Phe Val 435 440 445Lys Glu
Asn Pro Tyr Phe Thr Asn Arg Met Ser Val Asn Asn Gln Pro 450
455 460Phe Lys Ser Glu Asn Asp Leu Ser Tyr Tyr Lys
Val Tyr Gly Leu Leu465 470 475
480Asp Gln Asn Ile Leu Glu Leu Tyr Phe Asn Asp Gly Asp Val Val Ser
485 490 495Thr Asn Thr Tyr
Phe Met Thr Thr Gly Asn Ala Leu Gly Ser Val Asn 500
505 510Met Thr Thr Gly Val Asp Asn Leu Phe Tyr Ile
Asp Lys Phe Gln Val 515 520 525Arg
Glu Val Lys 53016581PRTSchizosaccharomyces pombe 16Met Phe Leu Lys Tyr
Ile Leu Ala Ser Gly Ile Cys Leu Val Ser Leu1 5
10 15Leu Ser Ser Thr Asn Ala Ala Pro Arg His Leu
Tyr Val Lys Arg Tyr 20 25
30Pro Val Ile Tyr Asn Ala Ser Asn Ile Thr Glu Val Ser Asn Ser Thr
35 40 45Thr Val Pro Pro Pro Pro Phe Val
Asn Thr Thr Ala Pro Asn Gly Thr 50 55
60Cys Leu Gly Asn Tyr Asn Glu Tyr Leu Pro Ser Gly Tyr Tyr Asn Ala65
70 75 80Thr Asp Arg Pro Lys
Ile His Phe Thr Pro Ser Ser Gly Phe Met Asn 85
90 95Asp Pro Asn Gly Leu Val Tyr Thr Gly Gly Val
Tyr His Met Phe Phe 100 105
110Gln Tyr Ser Pro Lys Thr Leu Thr Ala Gly Glu Val His Trp Gly His
115 120 125Thr Val Ser Lys Asp Leu Ile
His Trp Glu Asn Tyr Pro Ile Ala Ile 130 135
140Tyr Pro Asp Glu His Glu Asn Gly Val Leu Ser Leu Pro Phe Ser
Gly145 150 155 160Ser Ala
Val Val Asp Val His Asn Ser Ser Gly Leu Phe Ser Asn Asp
165 170 175Thr Ile Pro Glu Glu Arg Ile
Val Leu Ile Tyr Thr Asp His Trp Thr 180 185
190Gly Val Ala Glu Arg Gln Ala Ile Ala Tyr Thr Thr Asp Gly
Gly Tyr 195 200 205Thr Phe Lys Lys
Tyr Ser Gly Asn Pro Val Leu Asp Ile Asn Ser Leu 210
215 220Gln Phe Arg Asp Pro Lys Val Ile Trp Asp Phe Asp
Ala Asn Arg Trp225 230 235
240Val Met Ile Val Ala Met Ser Gln Asn Tyr Gly Ile Ala Phe Tyr Ser
245 250 255Ser Tyr Asp Leu Ile
His Trp Thr Glu Leu Ser Val Phe Ser Thr Ser 260
265 270Gly Tyr Leu Gly Leu Gln Tyr Glu Cys Pro Gly Met
Ala Arg Val Pro 275 280 285Val Glu
Gly Thr Asp Glu Tyr Lys Trp Val Leu Phe Ile Ser Ile Asn 290
295 300Pro Gly Ala Pro Leu Gly Gly Ser Val Val Gln
Tyr Phe Val Gly Asp305 310 315
320Trp Asn Gly Thr Asn Phe Val Pro Asp Asp Gly Gln Thr Arg Phe Val
325 330 335Asp Leu Gly Lys
Asp Phe Tyr Ala Ser Ala Leu Tyr His Ser Ser Ser 340
345 350Ala Asn Ala Asp Val Ile Gly Val Gly Trp Ala
Ser Asn Trp Gln Tyr 355 360 365Thr
Asn Gln Ala Pro Thr Gln Val Phe Arg Ser Ala Met Thr Val Ala 370
375 380Arg Lys Phe Thr Leu Arg Asp Val Pro Gln
Asn Pro Met Thr Asn Leu385 390 395
400Thr Ser Leu Ile Gln Thr Pro Leu Asn Val Ser Leu Leu Arg Asp
Glu 405 410 415Thr Leu Phe
Thr Ala Pro Val Ile Asn Ser Ser Ser Ser Leu Ser Gly 420
425 430Ser Pro Ile Thr Leu Pro Ser Asn Thr Ala
Phe Glu Phe Asn Val Thr 435 440
445Leu Ser Ile Asn Tyr Thr Glu Gly Cys Thr Thr Gly Tyr Cys Leu Gly 450
455 460Arg Ile Ile Ile Asp Ser Asp Asp
Pro Tyr Arg Leu Gln Ser Ile Ser465 470
475 480Val Asp Val Asp Phe Ala Ala Ser Thr Leu Val Ile
Asn Arg Ala Lys 485 490
495Ala Gln Met Gly Trp Phe Asn Ser Leu Phe Thr Pro Ser Phe Ala Asn
500 505 510Asp Ile Tyr Ile Tyr Gly
Asn Val Thr Leu Tyr Gly Ile Val Asp Asn 515 520
525Gly Leu Leu Glu Leu Tyr Val Asn Asn Gly Glu Lys Thr Tyr
Thr Asn 530 535 540Asp Phe Phe Phe Leu
Gln Gly Ala Thr Pro Gly Gln Ile Ser Phe Ala545 550
555 560Ala Phe Gln Gly Val Ser Phe Asn Asn Val
Thr Val Thr Pro Leu Lys 565 570
575Thr Ile Trp Asn Cys 58017627PRTAspergillus niger 17Met
His Ile Leu Pro Gly Ser Gln His Ala Ala Glu Leu Asp Asn Ser1
5 10 15Gly Thr Leu Ile His Ser Val
His Cys Asp Pro Glu Gln Lys Ala Lys 20 25
30Asn Ile Pro Gln Ser Thr Gly Ile Ala Gln Ala Ser Ser Glu
Trp Arg 35 40 45Pro Ser Tyr His
Leu Ala Ala Pro Arg Gly Trp Met Asn Asp Pro Cys 50 55
60Gly Leu Gly Tyr Asp Pro Thr Thr Gly Leu Tyr His Leu
Ser Phe Gln65 70 75
80Trp Asn Pro His Gly Asn Asp Trp Gly Asn Ile Ser Trp Gly His Ala
85 90 95Thr Ser Ser Asp Leu Val
Ser Trp Gln Ile Ser Pro Glu Pro Cys Leu 100
105 110Thr Pro Ser Ala Glu Tyr Asp Arg Cys Gly Val Phe
Thr Gly Cys Phe 115 120 125Arg Ser
His Gly Pro Asp Gly Lys Pro Gly Val Leu Thr Tyr Val Tyr 130
135 140Thr Ser Val Asn His Leu Pro Leu His Tyr Thr
Leu Pro Tyr Val Lys145 150 155
160Gly Ser Glu Ser Leu Ser Ile Ala Val Ser Arg Asp His Gly Lys Thr
165 170 175Trp Gln Arg Ile
Asp Ser Asn Pro Ile His Pro Gly Ala Pro Ala Gly 180
185 190Leu Glu Val Thr Gly Trp Arg Asp Pro Tyr Leu
Asn Cys Trp Pro Ser 195 200 205Leu
Arg Ala Gln Arg Gln Gly Gly Val Ala Ser Pro Asp Leu Tyr Gly 210
215 220Phe Ile Ser Gly Gly Ile Ala Lys Glu Ser
Pro Thr Val Phe Val Tyr225 230 235
240Val Val Asn Pro Asp Asn Leu Thr Glu Trp Thr Tyr Ile Gly Pro
Leu 245 250 255Leu His Val
Gly Leu Asn Tyr Arg Pro Ser Arg Trp Ser Gly Asp Leu 260
265 270Gly Val Asn Trp Glu Val Ala Asn Phe Phe
Thr Leu Thr Asp Gly Gly 275 280
285Val Ser Arg Asp Ile Val Ile Phe Gly Ala Glu Gly Cys Leu Ser Cys 290
295 300Glu Val Gly Ser Lys Arg Val Pro
Arg Ser Leu Leu Trp Met Cys Ile305 310
315 320Asn Val Arg Pro Gly Leu Gln Ala Gln Ser Ser Gly
Glu Pro Leu Ala 325 330
335Asp Tyr Ser Phe Ser Gly Ile Phe Asp His Gly Cys Cys Tyr Ala Ala
340 345 350Asn Ser Phe Trp Asp Pro
Val Thr Glu Glu Tyr Val Val Tyr Cys Trp 355 360
365Ile Thr Glu Glu Asp Leu Pro Asp Arg Leu Arg His Arg Gln
Gly Trp 370 375 380Ser Gly Ile Met Ser
Leu Pro Arg Leu Val Arg Leu Val Thr Leu His385 390
395 400Asn Val Lys Arg Ala His Gln Ser Lys Leu
Glu Ser Ile Thr Ser Val 405 410
415Glu Ile Glu Arg His Ser Gln Gly Thr Gln Val Arg Thr Leu Ser Val
420 425 430Arg Pro Asp Pro Arg
Leu Asn Ile Leu Arg Thr Ser Ala Arg Glu Leu 435
440 445His Leu Ser Asn Val Gln Leu Gly Ser Val Ala His
Gln Pro Pro Ala 450 455 460Phe Leu Pro
Leu Arg Thr Ala Arg Trp Glu Met Thr Ala Thr Phe Val465
470 475 480Ile Gly Thr His Cys Ala Ala
Val Gly Leu Glu Ile Gly His Ser Pro 485
490 495Asp Phe His Gln Arg Thr Thr Leu Ser Trp Ile Pro
Tyr Asp Glu Thr 500 505 510Phe
Thr Ile Glu Arg Pro Pro Leu His Asp Ala Gly Ile Asn His Val 515
520 525Pro Glu Thr Ala Pro His Thr Leu Phe
Thr Phe Cys Asn Asn Glu Gly 530 535
540Glu Glu Val Thr Glu Pro Leu Gln Ile His Ala Tyr Phe Asp Ala Ser545
550 555 560Val Leu Glu Val
Phe Val Asn Ser Arg Thr Val Ile Ser Thr Arg Ile 565
570 575Tyr Thr Pro His Ala Gln Val Cys Thr Gly
Leu Lys Phe Phe Ala Ser 580 585
590Ala Thr Glu Ser Gln Pro Lys Pro Ser Thr Ser Ala Pro Ala Ala Val
595 600 605Leu Val Arg Ala Asp Ile Trp
Asp Gly Leu Ser Val Ile Arg Asp Glu 610 615
620Ile Lys His625
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