# Patent

## Title
Genetically engineered yeast cell capable of producing lactate, method of producing the same, and method of producing lactate by using the cell

## Abstract

A genetically engineered yeast cell capable of producing lactate having increased TPI activity, a method of preparing the yeast cell, and a method of producing lactate by using the yeast cell.

## Background
RELATED APPLICATIONS
This application claims the benefit of Korean Patent Application No. 10-2013-0138359, filed on Nov. 14, 2013, in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.
INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 68,510 Byte ASCII (Text) file named 716720_ST25.TXT-Revised created on Feb. 2, 2015.
BACKGROUND
1. Field
The present disclosure relates to a genetically engineered yeast cell capable of producing lactate, a method of producing the same, and a method of producing lactate by using the cell.
2. Description of the Related Art
Lactate is an organic acid that is broadly used in various industrial fields, such as food, pharmaceutics, chemicals, and electronics. Lactate is colorless, odorless, and a low-volatile material that dissolves well in water. Lactate is non-toxic to the human body and thus may be used as a flavor agent, a taste agent, or a preserving agent. Also, lactate is an environment-friendly alternative polymer material and a raw material of a polylactic acid (PLA) that is biodegradable plastic.
PLA is a polyester-based resin that is obtained by ring opening polymerization (ROP) of lactide, a dimer which has been converted from lactic acid. PLA may be variously processed to a film, sheet, fiber, plastic, etc. Thus, demands for PLA as bioplastic have recently increased to broadly replace conventional typical petrochemical plastic, such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), or polystyrene (PS).
Lactate includes both a hydroxyl group and a carboxyl group and thus is highly reactive. Accordingly, lactate is easily converted into industrially important compounds, such as lactate ester, acetaldehyde, or propylene glycol, and thus has received attention as an alternative chemical material of the next generation in chemical industry.
Currently, lactate is produced by an industrially petrochemical synthesis process and a biotechnological fermentation process. The petrochemical synthesis process is performed by oxidizing ethylene derived from crude oil, preparing lactonitrile through addition of hydrogen cyanide after acetaldehyde, purifying by distillation, and hydrolyzing by using chloric acid or phosphoric acid. The biotechnological fermentation process is used to manufacture lactate from a reproducible carbon hydrate, such as, starch, sucrose, maltose, glucose, fructose, or xylose, as a substrate.
Therefore, a strain for efficiently producing lactate and a lactate production method using the strain are needed.
SUMMARY
Provided is a genetically engineered yeast cell capable of producing lactate, wherein the genetically engineered yeast cell has increased triose-phosphate isomerase (TPI) activity as compared to a yeast cell of the same species that is not genetically engineered.
Also provided is a method of producing the genetically engineered yeast cell by increasing the TPI activity of a yeast cell.
Further provided is a method of producing lactate by culturing the genetically engineered yeast cell in an appropriate medium and collecting the lactate from the culture.
Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic illustrating a p416-CCW12p-LDH vector;
FIG. 2 is a schematic illustrating a pUC57-ura3HA (Genetics 116: 541-545, August, 1987) vector;
FIG. 3 is a schematic illustrating a pUC57-ura3HA-ccw12p-LDH vector;
FIG. 4 is a schematic illustrating a p416TEF-S.TPI1 vector;
FIG. 5 is a schematic illustrating a p416TEF-T.TPI1 vector;
FIG. 6 illustrates a p416TEF-R.TPI1 vector;
FIG. 7 is a graph illustrating amount of lactate produced (g/L) and lactate production yield (%) from genetically engineered Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP compared to Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP that is not genetically engineered when polynucleotides encoding triose-phosphate isomerase (TPI) derived from Saccharomyces cerevisiae, TPI derived from Trypanosoma brucei, and TPI derived from a rabbit muscle cell are individually introduced to Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP.



DETAILED DESCRIPTION
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
As used herein, the term “lactate” denotes a lactic acid or a salt thereof.
As used herein, the term “genetically engineered” or “genetically modified” denotes a subject (e.g., cell) having an genetic composition (genotype) that is different from a naturally occurring (wild-type) cell of the same type, the genetic composition having been altered by genetic engineering techniques. The term “genetically engineered” may include “recombinant”. Recombination may include introducing exogenous nucleic acid with or without using a vehicle, such as a vector.
The genetically engineered yeast cell may be a recombinant yeast, which may include at least one recombinant nucleic acid or a recombinant protein. The recombinant yeast may include an expression vector or a cloning vector or may be genetically engineered to integrate a cloned nucleic acid sequence into the endogenous genome of the host cell. The genetic engineering includes gene modification, and the gene modification may be related to transcription, translation, and post-translation modification generating change in an enzyme's activity and/or selectivity under a selected and/or confirmed culture condition, and/or providing additional polynucleotides to increase the copy number and/or to introduce a recombinant of an enzyme in relation of lactate production.
As used herein, the terms “activity increase”, “enzyme activity increase”, “increased activity”, or “increased enzyme activity” denote that a cell or an isolated polypeptide or enzyme has an increased activity level compared to an activity level of a comparable cell of the same type or the original, unmodified enzyme or polypeptide. The activity may be increased by any amount. For instance, an enzyme conversion activity from a substrate to a product with respect to a corresponding enzyme may be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, or at least about 100% increased compared to the same biochemical conversion activity of an originally uncontrolled enzyme. A cell having an increased activity of an enzyme may be confirmed by using any method commonly known in the art such as high pressure liquid chromatography (HPLC).
As used herein, “inactivated” or “reduced” activity of a cell, an enzyme or a polypeptide, denotes a cell, an enzyme, or a polypeptide having an activity level that is lower than an activity level measured in a parent yeast cell having the original, unmodified enzyme or polypeptide. This includes a cell, an enzyme or a polypeptide having no activity. The activity may be reduced by any amount. For instance, an enzyme's conversion activity of a substrate to a product with respect to a corresponding enzyme may be reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% in comparison to the conversion activity of an enzyme that has not undergone a subject modification, e.g., a deletion or disruption mutation. The cells having reduced activity of the enzyme may be confirmed by using a commonly known method in the art. The inactivation or reduction includes the cases where a gene encoding an enzyme is not expressed or also where a gene encoding an enzyme has a lower level of expression compared to the expression level of the gene that has not undergone a subject modification, e.g., a deletion or disruption mutation, even when the enzyme is expressed or when an activity of the enzyme is removed or reduced.
An activity of the enzyme may be inactivated or reduced due to substitution, addition, or deletion of a part or a whole gene encoding the enzyme (e.g., a substitution, addition, or deletion mutation). For example, inactivation or reduction of the enzyme may be caused by homologous recombination or may be performed by transforming a vector including a part of sequence of the gene to the cell, culturing the cell so that the sequence may homogonously recombined with an endogenous gene of the cell, and then selecting cells, in which homologous recombination occurred, using a selection marker.
As used herein, the term “sequence identity” of a nucleic acid or a polypeptide refers to a degree of similarity of base groups or amino acid residues between two aligned sequences, when the two sequences are aligned to match each other as possible, at corresponding positions. The sequence identity is a value that is measured by aligning to an optimum state and comparing the two sequences at a particular comparing region, wherein a part of the sequence within the particular comparing region may be added or deleted compared to a reference sequence. A sequence identity percentage may be calculated, for example, by 1) comparing the two sequences aligned within the whole comparing region to an optimum 2) obtaining the number of matched locations by determining the number of locations represented by the same amino acids of nucleic acids in both of the sequences, 3) dividing the number of the matched locations by the total number of the locations within the comparing region (i.e., a range size), and 4) obtaining a percentage of the sequence identity by multiplying 100 to the result. The sequence identity percent may be determined by using a common sequence comparing program, for example, BLASTN or BLASTP (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc).
The term “parent cell” or “parent yeast cell” or the like refers to an original cell, for example, a non-engineered cell of the same type as an engineered yeast cell. With respect to a particular genetic modification, the “parent cell” can be a cell that lacks the particular genetic modification, but is identical in all other respects. Thus, a parent cell can be a cell used as starting material to produce a genetically engineered yeast cell having an activated or increased activity of a given protein (e.g., a protein having a sequence identity of about 95% or more to a triose-phosphate isomerase).
In confirming many different polypeptides having the same or similar function or activity or polynucleotides encoding polypeptides having the same or similar function or activity, sequence identities of several values may be used. For example, the sequence identities may include about 50% or greater, about 55% or greater, about 60% or greater, about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, about 95% or greater, about 96% or greater, about 97% or greater, about 98% or greater, about 99% or greater, or 100%.
The genetically engineered yeast cell may have a lactate-producing capability. The yeast cell may produce lactate at a percent yield of 34% or greater, for example, from 34 to 45%, 34 to 41%, from 35 to 41%, from 36 to 41%, from 37 to 41%, from 38 to 41%, from 38.5 to 40.5%, or from 39 to 40.5% with respect to glucose, i.e. the amount of lactate produced by the yeast cell divided by the glucose consumed by the yeast cell multiplied by 100.
Also, the lactate productivity of the genetically engineered cell may have a percent yield which is 9% or more, for example, from 9 to 25%, from 10 to 25%, from 11 to 25%, from 12 to 25%, from 13 to 25%, from 14 to 25%, from 15 to 25%, from 16 to 25%, from 17 to 25%, from 18 to 25%, from 19 to 25%, from 15 to 20%, from 16 to 20%, from 17 to 20%, from 18 to 20%, or from 19 to 20% increased compared to the lactate productivity of a cell that is not genetically engineered. The genetic engineering may increase an activity of polypeptide of triose-phosphate isomerase (TPI).
As used herein, the cell that is not genetically engineered includes a mother cell or a wild-type cell of the same species from which the yeast cell with an increased activity of TPI is derived.
As used herein, the term “triose-phosphate isomerase (TPI)” denotes an enzyme that catalyzes reversible conversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P). The TPI may be a polypeptide derived from EC 5.3.1.1.
In regard to the genetically engineered yeast cell with an increased TPI activity compared to that of a parent yeast cell, the TPI activity may be increased due to an increase in a copy number of an endogenous gene encoding TPI, or due to the introduction of an exogenous TPI gene, for instance, a TPI gene having increased activity compared to an endogenous TPI gene. The increase in copy number of the gene may be caused by introduction of exogenous TPI gene or genes, or by amplification of the endogenous gene encoding TPI. The gene encoding TPI may include an expression-regulation sequence of the gene.
The gene encoding TPI may be a heterologous gene. The term “heterologous gene” or “heterologous polynucleotide” indicates a nucleic acid or polypeptide that is not naturally found in a given host cell. The gene encoding TPI may be also a homologous gene. The homologous gene may refer to a gene from the same source as that of the recipient cell (e.g., Saccharomyces genus).
The term “heterologous” is understood as including the meaning of the term “exogenous”, and the term “exogenous” is commonly used in the art. The heterologous gene may refer to a gene from a source different than that of the recipient cell. The nucleic acid sequence encoding an enzyme is heterologous regardless of whether the heterologous nucleic acid sequence is integrated into the genome or not. In some embodiments, the increase in TPI activity may be caused by introduction of the heterologous gene encoding TPI.
Also, the increase in TPI activity may be caused by modification of the expression-regulation sequence of the gene. The regulation sequence may be a gene encoding a motif that may affect the gene expression. The gene expression may include both transcription and translation and may be understood as including protein production. Examples of the motif that may affect the gene expression may include a secondary-structure stabilization motif, a region having increased self-homology, a region having increased homology with respect to a natural gene, an RNA destabilization motif, a splice-activation motif, a polyadenylation motif, an adenine-rich sequence, and an endonuclease recognizing site.
The regulation sequence may be an appropriate promoter sequence, which is a nucleotide sequence that is recognized by a host cell for expression of the gene encoding TPI. The promoter sequence may include a transcription regulation sequence mediating expression of polypeptide. The promoter may be any nucleotide sequence that shows transcription activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
The regulation sequence may also include an appropriate transcription terminator sequence which is recognized by a host cell to terminate the transcription. The terminator sequence may be operably linked to a 3′ terminal of a nucleotide sequence encoding a polypeptide. The terminator sequence may be an arbitrary terminator functioning in a yeast cell. Also, a regulation sequence that regulates expression of a polypeptide in regard of growth of the host cell may be added. An example of a control system may be a system that turns on or off expression of the gene in response to a chemical or physical stimulus caused by presence of a control compound (i.e. an inducible expression system).
Also, the increase in TPI activity may be due to modification caused by mutation of TPI polypeptide.
The TPI-encoding gene may be derived from bacteria, yeast, fungus, or mammals. The yeast may include a Saccharomyces genus. The mammals may include a rabbit. The rabbit may be European rabbit (Oryctolagus cuniculus) or cottontail rabbit (genus Sylvilagus). The TPI may be derived from Saccharomyces cerevisiae, Trypanosoma brucei, a rabbit, or a combination thereof. The TPI derived from Saccharomyces cerevisiae may be one of proteins shown in Table 1. The TPI derived from the rabbit muscle cell may be one of proteins shown in Table 3.







TABLE 1







TPI derived from Saccharomyces cerevisiae and its gene









SEQ




ID NO
strain
gene





1
strain ATCC 204508/S288c
TPI1 YDR050C




YD9609.05C



strain AWRI1631
AWRI1631_42730



strain AWRI796
AWRI796_0798



strain FostersB
FOSTERSB_0776



strain JAY291
TPI1 C1Q_03637



strain Kyokai no. 7/NBRC 101557
K7_TPI1 SYK7_012011



strain Lalvin EC1118/Prise de
EC1118_1D0_2949g



mousse



strain Lalvin QA23
QA23_0786



strain RM11-1a
SCRG_00464



strain VIN 13
VIN13_0785



strain YJM789
TPI1 SCY_0954



Saccharomyces kudriavzevii VIN7
VIN7_0778


2
strain FostersO
FOSTERSO_0623


3
strain Zymaflore VL3
VL3_0789


4
Saccharomyces kudriavzevii VIN7
VIN7_6169
















TABLE 2







TPI derived from Trypanosoma brucei gambiense and its gene









SEQ




ID NO
strain
Gene





5
T. brucei brucei
Tb11.02.3210



(strain 927/4 GUTat10.1)



T. brucei gambiense
TbgDal_XI6230



(strain MHOM/CI/86/DAL972)



T. brucei gambiense
TbgDal_IX5650



(strain MHOM/CI/86/DAL972)


6
T. brucei brucei


7
T. brucei brucei
Tb09.211.1370



(strain 927/4 GUTat10.1)
















TABLE 3







TPI derived a rabbit muscle cell and its gene









SEQ ID NO
strain
Gene





8
Oryctolagus cuniculus (Rabbit)
TPI1


9
Oryctolagus cuniculus (Rabbit)
LOC100348316









The TPI may have an amino acid sequence having about 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of sequence identity with one amino acid sequence selected from among SEQ ID NO: 1 to 9. The gene encoding TPI may be one of genes shown in Tables 1 to 3. The gene encoding TPI may have one polynucleotide sequence selected from among SEQ ID NOS: 10 to 13.
Also, the yeast cell may be a mutated yeast cell for producing lactate in addition to a wild yeast cell. The mutated yeast cell may include other mutations, such as a mutation that renders the yeast cell resistant to an acid such as lactic acid, or lactate. Also, the mutated yeast cell may further include a natural LDH gene.
The yeast cell may be ascomycota. The ascomycota may be saccharomycetaceae. The saccharomycetaceae may be Saccharomyces genus, Kluyveromyces genus, Candida genus, Pichia genus, Issatchenkia genus, Debaryomyces genus, Zygosaccharomyces genus, or Saccharomycopsis genus. The Saccharomyces genus may be, for example, S. cerevisiae, S. bayanus, S. boulardii, S. bulderi, S. cariocanus, S. cariocus, S. chevalieri, S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, S. florentinus, S. kluyveri, S. martiniae, S. monacensis, S. norbensis, S. paradoxus, S. pastorianus, S. spencerorum, S. turicensis, S. unisporus, S. uvarum, or S. zonatus. The Kluyveromyces genus may be Kluyveromyces thermotolerans. The Candida genus may be Candida glabrata. The Zygosaccharomyces genus may be Zygosaccharomyces bailli or Zygosaccharomyces rouxii. 
The genetically engineered yeast cell may also be modified such that the activity of a polypeptide converting pyruvate to acetaldehyde, a polypeptide converting lactate to pyruvate, a polypeptide converting dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, or a combination thereof may be inactivated or decreased. Activity of a polypeptide may be inactivated or decreased due to deletion or disruption of a gene encoding the polypeptide. As used herein, the “deletion” or “disruption” of the gene includes mutation or deletion of the gene or a regulatory region of the gene (e.g., operator, promoter or terminator regions of the gene), or a part thereof, sufficient to disrupt or delete gene function or the expression of a functional gene product. Mutations include substitutions, additions, and deletions of one or more bases in the gene or its regulator regions. As a result, the gene is not expressed or has a reduced amount of expression, or the activity of the encoded protein or enzyme is reduced or eliminated. The deletion or disruption of the gene may be accomplished by any suitable genetic engineering technique, such as homologous recombination, mutation induction, or directed molecular evolution. When a cell includes a plurality of copies of the same gene or at least two different polypeptide paralogs, at least one gene may be deleted or disrupted.
In the yeast cell, an activity of a polypeptide converting pyruvate to acetaldehyde may be inactivated or decreased. The polypeptide converting pyruvate to acetaldehyde may be an enzyme which is classified to EC 4.1.1.1. The enzyme may be a pyruvate decarboxylase. For example, the enzyme may be PDC1, PDC2, PDC3, PDC4, PDC5 or PDC6. The polypeptide converting pyruvate to acetaldehyde may have an amino acid sequence having about 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of sequence identity with an amino acid sequence of SEQ ID NO: 14. The gene encoding the polypeptide converting pyruvate to acetaldehyde may have a nucleotide sequence of SEQ ID NO: 15. The gene may be a pdc gene encoding a pyruvate decarboxylase (PDC).
In the yeast cell, an activity of the polypeptide converting pyruvate to lactate may be inactivated or decreased. The polypeptide converting pyruvate to lactate may be a cytochrome c-dependent enzyme. The polypeptide converting pyruvate to lactate may be a lactate cytochrome c-oxidoreductase. The lactate cytochrome c-oxidoreductase may be an enzyme that is classified to EC 1.1.2.4 working on D-lactate or an enzyme that is classified to EC 1.1.2.3 working on L-lactate. The enzyme may be Cyb2. The polypeptide converting lactate to pyruvate may have about 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of sequence identity with an amino acid sequence of SEQ ID NO: 16. The gene encoding the polypeptide converting lactate to pyruvate may have a nucleotide sequence of SEQ ID NO: 17.
In the yeast cell, an activity of the polypeptide converting DHAP to glycerol-3-phosphate may be inactivated or decreased. The polypeptide converting DHAP to glycerol-3-phosphate may be a cytosolic glycerol-3-phosphate dehydrogenase. The enzyme may catalyze reduction of DHAP to glycerol-3-phosphate by using oxidation of NADH to NAD+. The enzyme may belong to EC 1.1.1.8 and may be GPD1 or GPD2. The GPD1 may have an amino acid sequence having about 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% or sequence identity with an amino acid sequence of SEQ ID NO: 18. The gene encoding GPD1 may have a nucleotide sequence of SEQ ID NO: 19.
In the yeast cell, an activity of converting pyruvate to lactate may be increased. The activity of converting pyruvate to lactate may be increased sufficiently enough to produce lactate.
The increase in activity of converting pyruvate to lactate may be caused by introduction of a polypeptide (or nucleic acid encoding the polypeptide) that converts pyruvate to lactate, or an increase in the expression of an existing nucleic acid encoding such a polypeptide. The increase in the expression may be caused by an increase in the copy number of a gene or by mutation of a control region of a gene. The increase in the copy number of a gene may be caused by amplification of an endogenous gene or by introduction of an exogenous gene. The mutation of the control region of the gene may be caused by mutation of a control region of an endogenous gene. The source of the exogenous gene may be a homogenous or heterogenous. The homogenous gene may be composed of elements that are all of the same kind as those of a recipient cell, whereas the heterogenous gene may be composed of elements that are of a different kind from those of a recipient cell.
The polypeptide converting pyruvate to lactate may be a lactate dehydrogenase. The lactate dehydrogenase may catalyze conversion of pyruvate to lactate. The lactate dehydrogenase may be an NAD(P)-dependent enzyme which may be L-lactate dehydrogenase or D-lactate dehydrogenase. The NAD(P)-dependent enzyme may be an enzyme that is classified to EC 1.1.1.27 which works on L-lactate or EC 1.1.1.28 which works on D-lactate.
The yeast cell may include a plurality of genes encoding a lactate dehydrogenase. The polynucleotide encoding lactate dehydrogenase may be derived from bacteria, yeasts, and fungus, mammals, or reptiles. The polynucleotide may be a polynucleotide that encodes at least one LDH selected from Pelodiscus sinensis japonicus, Ornithorhynchus anatinus, Tursiops truncatus, Rattus norvegicus, and Xenopus laevis LDH proteins. A lactate dehydrogenase derived from Pelodiscus sinensis japonicas, a lactate dehydrogenase derived from Ornithorhynchus anatinus, a lactate dehydrogenase derived from Tursiops truncatus, a lactate dehydrogenase derived from Rattus norvegicus, and a lactate dehydrogenase derived from Xenopus laevis may be, each respectively, have amino acid sequences having about 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of sequence identity with amino acid sequences of SEQ ID NO: 20, 21, 22, and 23. A gene that encodes the lactate dehydrogenase may have a nucleotide sequence of SEQ ID NO: 24.
The polynucleotide encoding LDH may be expressed from a vector including LDH derived from bacteria, yeast, fungus, mammals, or reptiles. The vector may include a replication origin, a promoter, a polynucleotide encoding a lactate dehydrogenase, and a terminator. The replication origin may include a yeast autonomous replication sequence (ARS). The yeast ARS may be stabilized by a yeast centrometric sequence (CEN). The promoter may be selected from the group consisting of a CYC promoter, a TEF promoter, a GPD promoter, and an ADH promoter. The CYC promoter, TEF promoter, GPD promoter, and ADH promoter may have, each respectively, nucleotide sequences of SEQ ID NO: 26, 27, 28, and 29. The terminator may be selected from the group consisting of phosphoglycerate kinase 1 (PGK1), cytochrome c transcription (CYC1), and GAL1. The CYC1 terminator may have a nucleotide sequence of SEQ ID NO: 30. The vector may further include a selection marker.
The polynucleotide encoding LDH may be included in a genome of a yeast cell. When the polynucleotide encoding LDH functions to produce active protein in a cell, the polynucleotide is deemed as “functional” in a cell. A yeast cell including the polynucleotide encoding the L-lactate dehydrogenase or the D-lactate dehydrogenase may produce a L-lactate enantiomer, a D-lactate enantiomer, or a salt thereof.
The yeast cell may include a polynucleotide encoding one LDH or a polynucleotide encoding multiple LDHs to 1 to 10 copies. The polynucleotide encoding the multiple LDHs may be a polynucleotide encoding, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 copies of LDHs. When the yeast cell includes the polynucleotide encoding multiple LDHs, the polynucleotide may include copies of the same polynucleotide or copies of polynucleotides encoding at least two different LDHs. The multiple copies of the polynucleotide encoding exogenous LDHs may be included in the same gene locus or multiple gene loci in a genome of a host cell. In this sense, a single “gene” may contain multiple copies of a polynucleotide encoding the same LDH or multiple copies of polynucleotides encoding at least two different LDHs.
Moreover, the yeast cell may be Saccharomyces cerevisiae, in which activities of a polypeptide encoding TPI and a polypeptide converting pyruvate to lactate are increased and activities of a polypeptide converting pyruvate to acetaldehyde, a polypeptide converting lactate to pyruvate, a polypeptide converting DHAP to glycerol-3-phosphate, and a combination thereof are inactivated or reduced. An expression vector including the polynucleotide encoding TPI for constructing a genetically engineered yeast cell capable of producing lactate, wherein the yeast cell is modified to have an increased copy number of genes encoding TPI or the increased expression compared to a cell that is not genetically engineered.
The polynucleotide may be operably linked to a regulation sequence appropriate for expressing the polynucleotide in an appropriate host. The regulation sequence may include a promoter, a terminator, or an enhancer. Also, the promoter may be operably linked with a sequence encoding a gene. As used here, the term “operably linked” denotes a functional connection between a nucleic acid expression-regulation sequence and another nucleotide sequence. In this regard, the regulation sequence may control transcription and/or translation of a nucleotide sequence encoding the gene.
The expression vector may be a yeast expression vector, a bacteriophage vector, or a cosmid vector. The yeast expression vector may be, for example, a vector for expression in Saccharomyces cerevisiae, and examples of the yeast expression vector may include pYepSec1, 2i, pAG-1, Yep6, Yep13, PEMBLYe23, pMFa, pJRY88, pYES2, and Saccharomyces cerevisiae/Escherichia coli shuttle vector, and pRS400-series vectors, such as a pRS416 vector.
In order to serve as an expression vector, the vector may include a replication origin, a promoter, a multiple cloning site (MCS), and a selection marker. The replication origin serves to allow a plasmid to have a replicating function independent from chromosomal replication in a host cell, the promoter works on the transcription process of an exogenous gene being inserted, the MCS allows the exogenous gene to be inserted through various restriction enzyme sites, and the selection marker serves to confirm whether the vector is properly introduced to the host cell. The selection marker includes an antibiotic-resistive gene, for example, a gene that is resistive to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, or tetracycline. Also, the selection marker may include an auxotrophic gene and may include, for example, a gene providing auxotrophy to one selected from uracil, tryptophan, leucine, and histidine.
Examples of the promoter appropriate for directing transcription of a nucleic acid in a yeast host cell including Saccharomyces genus include a PGK promoter, a GPD promoter, a PDC1 promoter, and a TEF1 promoter.
According to an aspect of the present disclosure, a genetically engineered yeast cell capable of producing lactate is provided, wherein the cell has an increased activity of triose-phosphate isomerase (TPI) compared to that of a parent cell.
According to another aspect of the present disclosure, a genetically engineered yeast cell capable of producing lactate is provided, wherein the yeast cell is modified to have an increased copy number of a gene encoding TPI or an increased expression of the gene compared to that of a parent cell.
According to another aspect of the present disclosure, a method of preparing a genetically engineered yeast cell capable of producing lactate includes introducing a polynucleotide encoding TPI to yeast is provided.
In the method, the genetically engineered yeast cell may be modified to have an increased copy number of a gene encoding TPI or the increased expression compared to a cell that is not genetically engineered. The increase in the copy number may be caused by introduction or amplification of the gene.
Also, the TPI may catalyze conversion of DHAP to glyceraldehyde-3-phosphate (GA3P).
According to another aspect of the present disclosure, a method of preparing the genetically engineered yeast cell is provided, the method including introducing a polynucleotide encoding TPI into a yeast cell. The descriptions of TPI and the genetically engineered yeast cell are as defined in the specification.
The method may include inactivating or decreasing activities of a pyruvate decarboxylase gene, a lactate cytochrome-c oxydoreductase, a glycerol-3-phosphate dehydrogenase gene, and a combination thereof. The descriptions of the pyruvate decarboxylase gene, lactate cytochrome-c oxydoreductase, and glycerol-3-phosphate dehydrogenase gene are as defined in the specification.
The method may also include introducing a polynucleotide encoding a lactate dehydrogenase into the yeast cell. The description of the lactate dehydrogenase is as defined above in the specification.
According to another aspect of the present disclosure, a method of preparing lactate using the genetically engineered yeast cell is provided. The method may include culturing the genetically engineered yeast cell in an appropriate medium; and collecting lactate from the culture. The description of the genetically engineered yeast cell is as defined above in the specification.
The culturing may be performed in a carbon source, for example, a medium containing glucose. The medium used in the culturing of a yeast cell may be a common medium suitable for growth of a host cell such as a minimal or composite medium containing appropriate supplements. A suitable medium may be purchased from commercial suppliers or may be prepared according to a known preparation method.
The medium used in the culturing may be a medium that satisfies particular conditions for growing a yeast cell. The medium may be one selected from the group consisting of a carbon source, a nitrogen source, a salt, trace elements, and a combination thereof.
The culturing condition for obtaining lactate from the genetically engineered yeast cell may be appropriately controlled. The culturing may be performed in an aerobic or anaerobic condition. For example, the yeast cell is cultured under an aerobic condition for its proliferation, and then, the yeast cell is cultured under an anaerobic condition to produce lactate. The anaerobic condition may include a dissolved oxygen (DO) concentration of 0% to 10%, for example, 0% to 8%, 0% to 6%, 0% to 4%, or 0% to 2%.
As used herein, the term “culture condition” denotes a condition for culturing a yeast cell. The culture condition may be, for example, a carbon source, nitrogen source, or oxygen condition for the yeast cell. The carbon source that is used by the yeast cell includes monosaccharides, disaccharides, or polysaccharides. In particular, glucose, fructose, mannose, or galactose may be used. The nitrogen source that is used by the yeast cell may be an organic nitrogen compound or an inorganic nitrogen compound. In particular, amino acid, amide, amine, nitrate, or ammonium salt may be used. An oxygen condition for culturing yeast cell may be an aerobic condition of a normal oxygen partial pressure, a low-oxygen condition including 0.1% to 10% of oxygen in the atmosphere, or an anaerobic condition including no oxygen. A metabolic pathway may be modified in accordance with a carbon source or nitrogen that may be actually used by a yeast cell.
Isolation of lactate from the culture may be performed by an isolation method commonly known in the art. The isolation method may be centrifugation, filtration, ion-exchange chromatography, or crystallization. For example, a supernatant obtained by centrifuging the culture at a low speed and removing a biomass may be separated through ion-exchange chromatography.
The method may further include polymerizing the lactate to form a polylactate polymer.
The present disclosure will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
Preparation of Strain for Highly-Efficient Production of Lactate and Preparation of Expression Vector
In order to block a production pathway of ethanol and glycerol as main byproducts by using Saccharomyces cerevisiae CEN.PK2-1D (S. cerevisiae CEN.PK2-1D (MATα ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8c; SUC2) EUROSCARF accession number: 30000B) as a lactate production strain, a pyruvate decarboxylase (pdc1) gene, which is a main enzyme of alcohol fermentation, a NAD-dependent glycerol-3-phosphate dehydrogenase (gpd1) gene, which is a main enzyme of glycerol biosynthesis, and a L-lactate cytochrome-c oxidoreductase2 (cyb2) gene, which is a lactate lyase, were deleted from the strain to be used as a strain for producing lactate. Also, in order to produce lactate, a lactate dehydrogenase (ldh) gene of SEQ ID NO: 24 was inserted to each of the removal locations while simultaneously removing the three genes, and thus 3 copies of lactate dehydrogenases were inserted to the strain.
The removal of each of the genes and the simultaneous insertion of the lactate dehydrogenase genes were performed by homologous recombination at an upstream part and a downstream part of target locus of an open reading frame of each of the genes to be removed or a region including a promoter and a terminator.
(1.1) Preparation of a L-LDH Overexpression Vector and a Gene Exchange Vector for Inactivating pdc1, gpd1, and Cyb2 Genes
(1.1.1) Preparation of a L-LDH Overexpression Vector
A CCW12 promoter PCR fragment obtained by performing Polymerase Chain Reaction (PCR) with a genomic DNA of Saccharomyces cerevisiae CEN.PK2-1D as a template and using primers of SEQ ID NO: 31 and SEQ ID NO: 32 was digested with SacI and XbaI, and the resultant was inserted into p416-GPD vector (ATCC 87360TM) digested with SacI and XbaI, producing p416-CCW12p vector.
Then, L-ldh gene (SEQ ID NO: 24) was amplified from Pelodiscus sinensis japonicus genomic DNA by PCR using primers of SEQ ID NO: 33 and SEQ ID NO: 34. The resulting L-ldh PCR fragment and p416-CCW12p obtained therefrom were digested with BamHI and SalI, and ligated, producing p416-CCW12p-LDH, which is a L-ldh expression vector.
The L-ldh expression vector has also a yeast autonomous replication sequence (ARS)/a yeast centrometric sequence (CEN) of SEQ ID NO: 25, a CYC promoter of SEQ ID NO: 26, a GPD promoter of SEQ ID NO: 27, and a CYC1 terminator of SEQ ID NO: 28. The L-ldh expression vector included a polynucleotide encoding L-ldh derived from Pelodiscus sinensis japonicus of SEQ ID NO: 24.
FIG. 1 is a view illustrating a p416-CCW12p-LDH vector. As shown in FIG. 1, the LDH derived from Pelodiscus sinensis japonicus was introduced into the vector.
(1.1.2) Preparation of a Gene Exchange Vector
PDC1, CYB2, and GPD1 genes were deleted by using a homologous recombination method, and at the same time, a gene exchange vector for introducing a L-LDH gene was prepared in the same manner described below. FIG. 2 illustrates a pUC57-ura3HA (Genetics 116: 541-545, August, 1987) vector. FIG. 3 illustrates a pUC57-ura3HA-CCW12p-LDH vector.
PCR was performed using the prepared p416-CCW12p-LDH as a template with primers of SEQ ID NOS: 35 and 36. The resulting PCR fragment and the prepared pUC57-ura3HA vector were digested with SacI and ligated, producing pUC57-ura3HA-CCW12p-LDH.
PCR was performed using the produced pUC57-ura3HA-CCW12p-LDH as a template with primers of SEQ ID NOS: 37 and 38, producing a PDC1 gene deletion cassette.
PCR was performed using the produced pUC57-ura3HA-CCW12p-LDH as a template with primers of SEQ ID NOS: 39 and 40, producing a CYB2 gene deletion cassette.
PCR was performed using the produced pUC57-ura3HA-CCW12p-LDH as a template with primers of SEQ ID NOS: 41 and 42, producing a GPD1 gene deletion cassette.
(1.2) Inactivation of pdc1, gpd1, and cyb2 Genes
A mutant strain of Saccharomyces cerevisiae CEN.PK2-1D, in which pdc1 is deleted, was produced in the same manner as follows. Saccharomyces cerevisiae CEN.PK2-1D was plated onto a YPD agar plate (including 10 g of yeast extract, 20 g of peptone, and 20 g of glucose) and incubated for 24 hours at 30° C., and then, a colony obtained therefrom was inoculated in about 10 ml of a YPD liquid medium and cultured for about 18 hours at about 30° C. The sufficiently grown culture solution was inoculated in about 50 ml of a YPD liquid medium contained in a 250 ml-flask at a concentration of 1% (v/v) and cultured in an incubator at a rate of about 230 rpm and at 30° C. After about 4 to 5 hours, when the optical density measured at 600 nanometers (OD600) reached about 0.5, the culture was centrifuged at a rate of about 4,500 rpm for about 10 minutes to harvest cells, and the cells were resuspended in a lithium acetate solution at a concentration of about 100 mM. Then, the cells were harvested by performing centrifugation at a rate of about 4,500 rpm for about 10 minutes, resuspended in a lithium acetate solution at a concentration of about 1 M including about 15% of glycerol, and then divided into 100 ul aliquots each.
In order to delete a pdc1 gene, the above yeast component cell and the PDC1 deletion cassette produced in Example 1.1.2 were mixed with 50% of polyethylene glycol and a single stranded carrier DNA and reacted in a water tub for about 1 hour at 42° C., and then, the culture solution was spread on a uracil-free minimal agar plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, and 1.4 g/L of amino acid dropout mix (-ura)) and grown for about 24 hours or more at 30° C. Ten colonies (mutant strains) grown on the plate were selected, patched onto the fresh uracil-free minimal agar plate, and at the same time, inoculated into a liquid medium including the same components contained in the uracil-free minimal agar plate to isolate the genomic DNA from the above mutant strains by using a commonly used kit (Gentra Puregene Cell kit, Qiagen, USA). In order to confirm deletion of pdc1 gene, PCR was performed using the isolated genomic DNA of the mutant strain as a template with primers of SEQ ID NOS: 43 and 44, and then, electrophoresis was performed on the obtained PCR product to confirm pdc1 deletion. As a result, Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh+ura3) was obtained.
Also, for additional gene deletion using the gene exchange vector, a selection marker URA3 gene, which was introduced for the preparation of a CEN.PK2-1D (Δ pdc1::ldh+ura3) strain, was removed from the strain. Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh+ura3) was inoculated in about 10 ml of a YPD liquid medium, cultured for about 18 hours at 30° C., and spread on a 5-FOA plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, 1.4 g/L of an amino acid dropout mix, and 1 μg/L of 5-fluoroorotic acid), and cultured for about 24 hours or more at 30° C. Ten colonies (a URA3 pop-out strain) grown on the 5-FOA plate were selected, patched onto the fresh 5-FOA plate, and, at the same time, cultured in a YPD liquid medium to isolate genomic DNA from the selected strain by using a commonly used kit (Gentra Puregene Cell kit, Qiagen, USA). In order to confirm deletion of URA3 gene by using the isolated genomic DNA of the URA3 pop-out strain as a template, PCR was performed using primers of SEQ ID NOS: 43 and 44, and then electrophoresis was performed on the obtained PCR product to confirm deletion of URA3 gene. As a result, Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh) was obtained.
Deletion of the cyb2 gene in Saccharomyces cerevisiae CEN.PK2-1D (Δpdc1::ldh) was prepared in the same manner, as follows. Saccharomyces cerevisiae CEN.PK2-1D (Δpdc1::ldh) was plated onto a YPD agar plate (including 10 g of yeast extract, 20 g of peptone, and 20 g of glucose) and incubated for 24 hours at 30° C., and then, a colony obtained therefrom was inoculated in about 10 ml of a YPD liquid medium and cultured for about 18 hours at 30° C. The sufficiently grown culture solution was inoculated in about 50 ml of a YPD liquid medium contained in a 250 ml-flask at a concentration of 1% (v/v) and cultured in an incubator at a rate of about 230 rpm and at 30° C. After about 4 to 5 hours, when the OD600 reached about 0.5, the culture was centrifuged at a rate of about 4,500 rpm for about 10 minutes to harvest cells, and the cells were resuspended in a lithium acetate solution at a concentration of about 100 mM. Then, the cells were harvested by performing centrifugation at a rate of about 4,500 rpm for about 10 minutes, resuspended in a lithium acetate solution at a concentration of about 1 M including about 15% of glycerol, and then divided into 100 ul aliquots each.
In order to delete a cyb2 gene, a cyb2 deletion cassette, which is prepared in Examples 1 and 2 in the same manner as the pdc1 deletion cassette was prepared in Example 1.1.2, and the above pdc1-deleted competent cell were mixed with 50% of polyethylene glycol and a single stranded carrier DNA and reacted in a water tub for about 1 hour at 42° C., and then, the culture solution was spread on a uracil-free minimal agar plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, and 1.4 g/L of amino acid dropout mix (-ura)) and grown for about 24 hours or more at 30° C. Ten colonies (mutant strains) grown on the plate were selected, patched onto the fresh uracil-free minimal agar plate, and at the same time, inoculated into a liquid medium including the same components contained in the uracil-free minimal agar plate to isolate the genomic DNA from the above mutant strains by using a commonly used kit (Gentra Puregene Cell kit, Qiagen, USA). In order to confirm deletion of cyb2 gene, PCR was performed using the isolated genomic DNA of the mutant stain as a template with using primers of SEQ ID NOS: 45 and 46, and then, electrophoresis was performed on the obtained PCR product to confirm cyb2 deletion. As a result, Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh Δ cyb2::ldh+ura3) was obtained.
Also, for additional gene deletion using the gene exchange vector, a URA3 gene as a selection marker of the cyb2 deletion cassette was removed by using the URA3 pop-out method as described above. Saccharomyces cerevisiae CEN.PK2-1D (Δpdc1::ldh Δcyb2::ldh+ura3) was inoculated in about 10 ml of a YPD liquid medium, cultured for about 18 hours at 30° C., and spread on a 5-FOA plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, 1.4 g/L of an amino acid dropout mix, and 1 μg/L of 5-fluoroorotic acid), and cultured for about 24 hours or more at 30° C. Ten colonies (a URA3 pop-out strain) grown on the 5-FOA plate were selected, patched onto the fresh 5-FOA plate, and, at the same time, cultured in a YPD liquid medium to isolate genomic DNA from the selected strain by using a commonly used kit (Gentra Puregene Cell kit, Qiagen, USA). In order to confirm deletion of URA3 gene by using the isolated genomic DNA of the URA3 pop-out strain as a template, PCR was performed using primers of SEQ ID NOS: 45 and 46, and then electrophoresis was performed on the obtained PCR product to confirm deletion of URA3 gene. As a result, Saccharomyces cerevisiae CEN.PK2-1D (Δpdc1::ldh Δcyb2::ldh) was obtained.
A gpd1 deletion strain of Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldhΔcyb2::ldh) was prepared in the same manner as follows. Saccharomyces cerevisiae CEN.PK2-1D (Δpdc1::ldhΔcyb2::ldh) was plated onto a YPD agar plate (including 10 g of yeast extract, 20 g of peptone, and 20 g of glucose) and cultured for about 24 hours at 30° C., and then, a colony obtained therefrom was inoculated in about 10 ml of a YPD liquid medium and cultured for about 18 hours at 30° C. The sufficiently grown culture solution was inoculated in about 50 ml of a YPD liquid medium contained in a 250 ml-flask at a concentration of 1% (v/v) and cultured in an incubator at a rate of about 230 rpm and a temperature of about 30° C. After about 4 to 5 hours, when the OD600 reached about 0.5, the culture was centrifuged at a rate of about 4,500 rpm for about 10 minutes to obtain cells, and the cells were resuspended in a lithium acetate solution at a concentration of about 100 mM. Then, the cells were obtained by performing centrifugation at a rate of about 4,500 rpm for about 10 minutes, resuspended in a lithium acetate solution at a concentration of about 1 M including about 15% of glycerol, and then divided into 100 ul aliquots each.
In order to delete a gpd1 gene, a gpd1 deletion cassette, which is prepared in Example 1.2 in the same manner as the pdc1 deletion cassette and the cyb2 deletion cassette were prepared, and the above pdc1 and cyb2-deleted competent cell were mixed with 50% of polyethylene glycol and a single stranded carrier DNA and reacted in a water tub for about 1 hour at 42° C., and then, the culture solution was plated onto a uracil-free minimal agar plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, and 1.4 g/L of amino acid dropout mix (-ura)) in a plate and cultured for 24 hours or more at 30° C. Ten colonies (a mutant strain) formed in the plate were selected, transferred to another uracil-free minimal agar plate, and, at the same time, cultured in a liquid medium including the same components contained in the uracil-free minimal medium to separate genome DNA from the strain by using a commonly used kit (Gentra Puregene Cell kit, Qiagen, USA). In order to confirm deletion of gpd1 by using the separated genome DNA of the mutant strain as a template, PCR was performed using primers of SEQ ID NOS: 47 and 48, and then, electrophoresis was performed on the obtained PCR product to confirm gpd1 deletion. As a result, Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh Δ cyb2::ldh Δ gpd1::ldh+ura3) was obtained.
Also, for additional gene deletion using the gene deletion vector, a URA3 gene as a selection marker of the gpd1 deletion cassette was removed by using the URA3 pop-out method as described above. Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh Δcyb2::ldh Δ gpd1::ldh+ura3) was inoculated in about 10 ml of a YPD liquid medium, cultured for about 18 hours at 30° C., plated onto a 5-FOA plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, 1.4 g/L of an amino acid dropout mix, and 1 μg/L of 5-fluoroorotic acid) in a plate, and cultured for about 24 hours or more at 30° C. Ten colonies (a URA3 pop-out strain) formed in the plate were selected, transferred to another 5-FOA plate, and, at the same time, cultured in a YPD liquid to separate genome DNA from the strain by using a commonly used kit (Gentra Puregene Cell kit, Qiagen, USA). In order to confirm deletion of URA3 by using the separated genome DNA of the URA3 pop-out strain as a template, PCR was performed using primers of SEQ ID NOS: 47 and 48, and then electrophoresis was performed on the obtained PCR product to confirm URA3 deletion. As a result, Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh Δcyb2::ldh Δ gpd1::ldh) was obtained.
Saccharomyces cerevisiae CEN.PK2-1D (Δ pdc1::ldh Δ cyb2::ldhΔgpd1::ldh) was deposited in Korean Collection for Type Cultures (KCTC) on May 30, 2013, and received an accession number KCTC 12415BP.
Example 2
Preparation of a Gene Expression Vector Encoding TPI 1 and Preparation of Saccharomyces cerevisiae Introduced with Gene Encoding TPI 1
In order to prepare a lactate production strain with an enhanced carbon flux in the corresponding process, TPI with a polypeptide activity converting DHAP to GA3P was overexpressed or TPI of an exogenous cell was introduced.
(2.1) Preparation of a Gene Expression Vector Encoding TPI 1
A gene encoding TPI 1 (S. TPI1) of SEQ ID NO: 10 was amplified by performing PCR using gDNA (genomic DNA) of Saccharomyces cerevisiae as a template and primer sequences of SEQ ID NOS: 49 and 50.
The resulting gene encoding TPI 1 derived from Saccharomyces cerevisiae was introduced to a p416 TEF plasmid (ATCC catalog #87368), which is a low copy number centromeric plasmid by using a restriction enzyme BamHI/SalI to prepare p416 TEF-S.TPI1. FIG. 4 is a view illustrating a p416TEF-S.TPI1 vector.
Also, a gene encoding TPI 1 was amplified by performing PCR using gDNA of Trypanosoma brucei as a template and primers of sequences of SEQ ID NOS: 51 and 52.
The resulting gene encoding TPI 1 derived from Trypanosoma brucei was introduced to a p416 TEF plasmid (ATCC catalog #87368), which is a low copy number centromeric plasmid by using a restriction enzyme BamHI/SalI to prepare p416 TEF-T.TPI1. FIG. 5 is a view illustrating a p416TEF-T.TPI1 vector.
Also, a gene encoding TPI 1(R. TPI1) of SEQ ID NO: 13 was amplified by performing PCR using gDNA of a rabbit muscle cell as a template and primers of sequences of SEQ ID NOS: 53 and 54.
The resulting gene encoding TPI 1 derived from a rabbit muscle cell was introduced to a p416 TEF plasmid (ATCC catalog #87368), which is a low copy number centromeric plasmid by using a restriction enzyme BamHI/SalI to prepare p416 TEF-R.TPI1. FIG. 6 is a view illustrating a p416TEF-R.TPI1 vector.
(2.2) Preparation of Saccharomyces cerevisiae Containing a Gene Encoding a Version of TPI1
The KCTC12415BP strain prepared in Example 2 was plated onto a YPD agar plate (including 10 g of yeast extract, 20 g of peptone, and 20 g of glucose) and incubated for 24 hours at 30° C., and then, a colony obtained therefrom was inoculated in about 10 ml of a YPD liquid medium and cultured for about 18 hours at 30° C. The sufficiently grown culture solution was inoculated in about 50 ml of a YPD liquid medium contained in a 250 ml-flask at a concentration of 1% (v/v) and cultured in an incubator at a rate of about 230 rpm and at 30° C.
After about 4 to 5 hours, when the OD600 reached about 0.5, the culture was centrifuged at a rate of about 4,500 rpm for about 10 minutes to obtain cells, and the cells were resuspended in a lithium acetate solution at a concentration of about 100 mM. Then, the cells were obtained by performing centrifugation at a rate of about 4,500 rpm for about 10 minutes, resuspended in a lithium acetate solution at a concentration of about 1 M including about 15% of glycerol, and then divided into 100 ul aliquots each.
Each of p416 TEF-S.TPI1, p416 TEF-T.TPI1, and p416 TEF-R.TPI1 prepared in Example 2.1 and the above competent cell were mixed with 50% of polyethylene glycol and a single stranded carrier DNA and reacted in a water tub for about 1 hour at 42° C., and then, the culture solution was spread on a uracil-free minimal agar plate (including YSD, 6.7 g/L of yeast nitrogen base without amino acids, and 1.4 g/L of amino acid dropout mix (-ura)) and grown for about 24 hours or more at 30° C. Ten colonies (mutant strains) grown on the plate were selected, patched onto the fresh YSD (-ura) agar plate, and at the same time inoculated into a liquid YSD (-ura) medium to confirm that the p416 TEF-S.TPI1, p416 TEF-T.TPI1, and p416 TEF-R.TPI1 were introduced into each of the strains.
Example 3
Lactate Production Using the Genetically Engineered S. cerevisiae 
The culturing process described below was performed to evaluate lactate product fermentation of the strain prepared in Example 2 and Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP as a control group.
The culture conditions including a first process and a second process below were formed in the liquid YSD (ura-), which is a uracil-free minimal liquid medium, in a 50 ml shaking flask.
The first process of the culture conditions was as follows. 50 ml of a YSD (ura-) liquid medium containing 4% glucose was cultured in a 250 ml shaking flask at a rate of about 250 rpm at about 30° C. The strain introduced with a TPI expression vector was inoculated into each of the shaking flasks, and the strain was grown until the strain reached a log-growth phase, i.e., about 15 hours to about 16 hours after the inoculation, or until the OD600 reached about 5 to 8.
The second process of the culture conditions was as follows. 50 ml of the culture solution was centrifuged at a rate of about 3000 rpm for about 10 minutes to remove the supernatant. Then, the strain was resuspended in a 50 ml of fresh YSD (ura-) medium containing 8% glucose having 50 ml of the final volume, and the culture was moved to a 250 ml flask and grown at a rate of 80 rpm at 30° C. The culturing was performed until all the glucose was consumed, i.e., for about 24 hours to about 30 hours. Metabolites of the strain including lactate were measured by using HPLC to obtain lactate production yields. The results are shown in Table 4.










TABLE 4







Amount of lactate



Origin of introduced TPI gene
OD600
production (g/L)
Yield (%)


















None
7.23
22
33.8


Saccharomyces cerevisiae
7.97
22.5
37.1


Trypanosoma brucei
8.32
25
38.9


Rabbit muscle cell
8.11
25.5
40.4









As shown in Table 4, the Saccharomyces cerevisiae strains overexpressing the TPI genes derived from Saccharomyces cerevisiae, Trypanosoma brucei, and a rabbit muscle cell showed high lactate productivity and high lactate yield compared to those of Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP in which TPI gene is not introduced. Degrees of the increases in the productivity and yield were measured by comparing the levels of lactate productivity and yield of a control strain (Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP). Yield of lactate was calculated as a percent of the amount of produced lactate divided by the amount of consumed glucose, and the results are shown in Table 4.
Also, the lactate productivity and yield of the Saccharomyces cerevisiae strains overexpressing the TPI genes derived from Saccharomyces cerevisiae, Trypanosoma brucei, and a rabbit muscle cell compared to the lactate productivity and yield of Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP are shown in FIG. 7 and Table 5. FIG. 7 is a view of lactate productivity and a lactate production yield (%) of genetically engineered Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP compared to Saccharomyces cerevisiae CEN.PK2-1D KCTC12415BP that is not genetically engineered when polynucleotides encoding TPI derived from Saccharomyces cerevisiae, TPI derived from Trypanosoma brucei, and TPI derived from a rabbit muscle cell are introduced to Saccharomyces cerevisiae CEN.PK2-1 D KCTC12415BP.











TABLE 5








Production





Increase
Yield



Origin of TPI gene
(%)
Increase (%)




















Saccharomyces cerevisiae
2.3
9.8



Trypanosoma brucei
13.6
15.1



Rabbit muscle
15
19.5










As shown in FIG. 1 and Table 5, S. cerevisiae strains overexpressing TPI gene derived from Saccharomyces cerevisiae, Trypanosoma brucei, and a rabbit muscle cell showed an increasingly high lactate productivity and lactate production yield, in that order.
[Accession Number]
Research Center Name: Korean Collection for Type Cultures (KTCT)
Accession Number: KCTC 12415BP
Accession Date: May 30, 2013
As described above, according to the one or more of the above embodiments of the present invention, a genetically engineered yeast cell may produce lactate in a high yield, a vector may be used in a method of preparing a yeast cell capable of producing lactate in a high yield, a yeast cell capable of producing lactate in a high yield may be prepared by using a method of preparing a microorganism, and lactate may be produced in a high yield by using a method of producing lactate.
It should be understood that the exemplary embodiments described therein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.
While one or more embodiments of the present invention have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A genetically engineered yeast cell capable of producing lactate, wherein the yeast cell has an exogenous gene encoding triose-phosphate isomerase (TPI) and an increased triose-phosphate isomerase (TPI) activity compared to a parent yeast cell, and the yeast cell further has an exogenous polynucleotide encoding a polypeptide that converts pyruvate to lactate and increased activity of converting pyruvate to lactate compared to a parent yeast cell.

2. The genetically engineered yeast cell of claim 1, wherein the yeast cell is Saccharomyces genus, Kluyveromyces genus, Candida genus, Pichia genus, Issatchenkia genus, Debaryomyces genus, Zygosaccharomyces genus, or Saccharomycopsis genus.

3. The genetically engineered yeast cell of claim 1, wherein the TPI gene is a homologous gene.

4. The genetically engineered yeast cell of claim 1, wherein the TPI gene is a heterologous gene.

5. The genetically engineered yeast cell of claim 1, wherein the TPI is from Saccharomyces cerevisiae, Trypanosoma brucei, or a rabbit.

6. The genetically engineered yeast cell of claim 1, wherein the TPI has 60% or more sequence identity with any one of SEQ ID NOS: 1 to 9.

7. The genetically engineered yeast cell of claim 1, wherein the gene encoding TPI comprises a nucleotide sequence selected from SEQ ID NOS: 10 to 13.

8. A method of producing lactate comprising
culturing the yeast cell of claim 1; and
collecting lactate from the culture.

9. A method of preparing a yeast cell of claim 1, wherein the method comprises introducing a TPI gene in to the yeast cell, thereby increasing the copy number of a TPI gene in the yeast cell.

10. The method of claim 9, wherein the TPI gene is a heterologous gene.

11. The method of claim 10, wherein the TPI gene is from Trypanosoma brucei, or a rabbit.

12. The method of claim 9, wherein the TPI gene is a homologous gene, and the gene is from Saccharomyces cerevisiae.

13. The genetically engineered yeast cell of claim 1, wherein the TPI has 90% or more sequence identity with any one of SEQ ID NOS: 1 to 9.

14. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell produces lactate at a percent yield of 34% or greater.