# Patent

## Title
Genetically engineered yeast cell with enhanced EDC activity and capability of producing lactate, method of producing the yeast cell, and method of producing lactate by using the yeast cell

## Abstract

A genetically engineered yeast cell with enhanced activity of an EDC protein compared to that of a parent cell and capability of producing lactate, a method of producing the yeast cell, and a method of producing lactate by using the yeast cell.

## Background
RELATED APPLICATION
This application claims the benefit of Korean Patent Application No. 10-2014-0119374, filed on Sep. 5, 2014, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety 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 70,134 Byte ASCII (Text) file named “719359_ST25.TXT-Revised” created on Nov. 11, 2015.
BACKGROUND
1. Field
The present disclosure relates to genetically engineered yeast cells capable of producing lactate, methods of producing the yeast cells, and methods of producing lactate by using the yeast cells.
2. Description of the Related Art
Lactate is an organic acid that is widely used in a variety of industrial fields, including the food, pharmaceutical, chemical, and electronic industries. Lactate is a colorless, odorless, water-soluble, low-volatile material. Lactate is not toxic to the human body, and thus is used as a flavoring agent, a souring agent, a preserving agent, or the like. Lactate is also used as a source of polylactic acid (PLA) that is an environmentally friendly, biodegradable plastic known as an alternate polymeric material. PLA is a polyester-based resin prepared for polymerization by conversion of lactic acid to its dimmer, i.e., lactide, and then, by ring-opening polymerization of the lactide. In this regard, PLA may be processed into a variety of forms, such as films, sheets, fibers, and injections. Accordingly, PLA has been increasingly demanded as a bioplastic material to replace the existing general-purpose petrochemical plastics, such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), and polystylene (PS). In addition, lactate has both a hydroxyl group and a carboxyl group, and thus has desirable reactivity. Accordingly, lactate is easily converted to industrially important compounds, such as lactate ester, acetaldehyde, or propylene glycol, and thus, in the chemical industry field, PLA also has attracted attention as a next-generation alternative chemical raw material.
In recent years, lactate is industrially produced by a petrochemical synthesis process and a biotechnological fermentation process. According to the petrochemical synthesis process, ethylene derived from crude is oxidized, and via acetaldehyde, lactonitrile is obtained by addition of hydrogen cyanide. Then, lactonitrile is purified by distillation and hydrolyzed by using hydrochloric acid or sulfuric acid, thereby producing lactate. According to the biotechnological fermentation process, renewable carbohydrates, such as starch, sucrose, maltose, glucose, fructose, and xylose, are used as substrates to produce lactate. Thus, in view of the art a strain capable of producing lactate in an effective manner and a method of producing lactate using said strain have been demanded. In order to meet these needs, a method of producing lactate by using a microorganism has been recently developed. However, when using a microorganism, due to its homeostasis, mass production of only one material is restricted, and thus, the present inventive concept is completed in the process of studying solutions for the problem.
SUMMARY
Provided is a genetically engineered yeast cell having increased EDC protein activity compared to the EDC protein activity of a parent cell thereof, wherein the yeast cell produces lactate. Provided is a method of producing a genetically engineered yeast cell that produces lactate, the method comprising overexpressing a polynucleotide that encodes an EDC protein in a yeast cell; and introducing a polynucleotide into the yeast cell that encodes a polypeptide that converts pyruvate to L-lactate.
Provided is a method of producing lactate, the method comprising:
culturing the genetically engineered yeast cell in a cell culture medium, whereby the yeast cell produces lactate.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects will become apparent and more readily appreciated from the following description of the exemplary embodiments, taken in conjunction with the accompanying drawings in which:
FIG. 1 is a vector map of a pCtB1 vector;
FIG. 2 is a vector map of a pCS-Ex1.1 vector; and
FIG. 3 is a vector map of a pCS-Ex1 vector.



DETAILED DESCRIPTION
Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present exemplary embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the exemplary embodiments are described below, by referring to the figures, merely to explain and illustrate aspects of the disclosure. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
The term “genetically engineered” and the like as used herein refers to a biological substance (e.g., cell, polypeptide, nucleic acid, etc.) that contains a non-natural genetic modification, or is produced as the result of a non-natural genetic modification. The non-natural genetic modification can be introduced by any suitable genetic engineering technique.
The terms “increase in activity” or “increased activity” and the like as used herein refers to an detectable increase in a biological activity (e.g., enzyme activity, expression level, etc.) of a cell, polypeptide, or nucleic acid. Increased activity includes, for instance, increases in the amount of a protein or enzyme sufficient to produce a detectable increase in the activity thereof. Increased activity also includes, for instance, increases in the specific activity of a protein or enzyme, which causes a detectable increase in activity even if the amount of protein or enzyme is unchanged. The term “increase in activity” or “increased activity” of a genetically modified (e.g., genetically engineered) cell, protein or enzyme can be any increase as compared to a cell, a protein, or a enzyme of the same type without the genetic modification (e.g., an original, parent, or “wild-type” cell, protein, or enzyme). The term “cell activity” may refer to activity of a specific protein or enzyme in a cell. The activity of a genetically modified or engineered cell, protein, or enzyme may be increased by any amount, for example, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more enhanced (increased) as compared to the activity of a non-engineered cell, protein, or enzyme, e.g., a wild-type or parent cell, protein, or enzyme, of the same type. Similarly, the activity of a specific protein or enzyme in a cell may be increased by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more compared to the activity of the same protein or enzyme in a parent cell, e.g., a non-engineered protein or enzyme of the same type in a cell. Cells having enhanced activity of a protein or enzyme may be identified by using any method commonly known in the art.
The term “copy number increase” includes an increase in copy number of a gene (e.g. a polynucleotide) by introduction of a gene into a cell (e.g., the introduction of an endogenous or an exogenous gene into a cell) and/or amplification of an endogenous gene. The introduction of an endogenous gene may be carried out using a vehicle such as a vector. The introduction of a gene into the cell may be a transient introduction, in which the gene is not integrated into the genome, or the introduction of the gene into the cell may result in integration into the genome. The introduction may occur by, for example, introducing a vector containing a polynucleotide that encodes a desired polypeptide into the cell, and replicating the vector in the cell or integrating the polynucleotide into a genome of a cell. The introduction of the vector into the cell may be performed using techniques that are well known to those of ordinary skill in the art, such as heat shock transformation, electroporation, and/or the exposure of the cell to divalent cations.
The polynucleotide having an increased copy number may be endogenous or exogenous. An endogenous gene refers to a gene present in a genetic material within a microorganism prior to a given genetic modification. An exogenous gene refers to a gene that is introduced into a host cell, such as a gene integrated into a genome of a host cell. The gene introduced into a host cell may be homologous or heterologous with respect to the host cell.
The term “heterologous” may denote that a gene is not a native gene, but a foreign gene, whereas “homologous” refers to a gene that is native to the host cell.
The term “gene” as used herein refers to a polynucleotide that is expressed by at least one of transcription and translation. An example of a gene is a nucleic acid fragment capable of being transcribed into mRNA or translated into a protein. A “gene” may or may not include a coding region or a regulatory sequence of a 5′-non coding sequence and a 3′-non coding sequence in addition to the coding region.
The term “cell”, “strain”, or “microorganism” as used herein may include bacteria, yeast, or fungi to be interchangeably used each other.
The term “decrease in activity” or “decreased activity” as used herein refers to any detectable decrease in a biological activity (e.g., enzyme activity, expression level, etc.) of a cell, polypeptide, or nucleic acid. Decreased activity includes, for instance, decreases in the amount of a protein or enzyme sufficient to produce a detectable increase in the activity thereof. Decreased activity also includes, for instance, decreases in the specific activity of a protein or enzyme, which causes a detectable decrease in activity even if the amount of protein or enzyme is unchanged. A decrease in activity of a genetically modified (genetically engineered) cell, protein, or enzyme can be any decrease as compared to a cell, protein, or enzyme without the genetic modification (e.g., a genetically non-engineered cell, such as a parent or “wild-type” cell, protein, or enzyme). The term “decrease in activity” or “decreased activity” refers to an isolated enzyme or polypeptide that has lower activity than an original or wild-type enzyme or polypeptide. The term “decrease in activity” or “decreased activity” also refers to complete elimination of activity. The activity of a genetically modified cell, protein, or enzyme (e.g., enzymatic activity in conversion of a substrate of a modified (e.g., genetically engineered) cell or enzyme to a product) may be reduced by any amount, such as 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% reduced compared to the activity of a non-engineered cell or enzyme, e.g., a parent or a wild-type cell, protein, or enzyme. The decrease in activity of an enzyme or cell may be identified by using any method commonly known in the art. The decrease in activity may include situations where an enzyme has no activity or reduced activity even when the enzyme is expressed as compared to a cell having a gene that is not modified, e.g., a parent cell or a wild-type cell, or a case where a gene encoding the enzyme is not expressed or an expression amount of the gene is decreased compared to an expression amount of an the enzyme in a non-genetically engineered cell.
The term “parent cell” as used herein may denote an original cell, for example, a non-engineered cell of the same type with respect to 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. In this regard, the parent cell may be a cell used as a starting material to produce a genetically engineered yeast cell having enhanced activity of a given protein (e.g., a protein having 95% or more sequence identity with an EDC enzyme).
With regard to a subject cell having a particular genetic modification, a parent cell is identical to the subject cell with the exception of the genetic modification, and accordingly, may be a reference cell with respect to the genetically modified cell. The term “genetic modification” refers to an artificially modified configuration or structure of a genetic material (e.g., an exogenous protein, a mutation in a polynucleotide, etc.) of a cell. The parent cell may be a cell without the subject genetic modification, e.g., a cell that is not genetically engineered to have enhanced activity of EDC. The parent cell may be a parent yeast cell.
The term “wild-type” polypeptide or polynucleotide refers to a polypeptide or polynucleotide without specific genetic modification as might be found in nature.
The term “disruption” as used herein may denote a genetic modification (e.g., a mutation, a substitution, or a deletion of one or more nucleotides or a gene) that causes a decrease in the expression of a referenced gene. The term “disruption” may include a case where the referenced gene is genetically engineered to have no expression (hereinafter, referred to as “inactivation” of a gene), or a case where the referenced gene is genetically engineered to be expressed at a reduced expression amount (hereinafter, referred to as “attenuation” of a gene). The inactivation of the gene may include not only situations where a functional product of a gene is not expressed, but also situations where a gene is expressed without the expression of the functional product (e.g., expression of a non-functional gene product). The attenuation of the gene may denote a decrease in expression of a gene, or a decrease in function of the expressed gene product (e.g., expression of a gene product with reduced function). That is, the attenuation of the gene may denote a decrease in the functional product even if the expression of the gene itself is increased. The functional product of the gene may refer to a biochemical activity or physiological function (e.g., enzyme activity) that a product of a gene (e.g., an enzyme) has in a parent cell or a wild-type cell. Therefore, the disruption may include functional disruption of the gene. The genetic modification may include: modification by introduction of a polynucleotide encoding a polypeptide; substitution, addition, insertion, or deletion of at least one nucleotide of a gene (polynucleotide); or chemical mutation of genetic material. The genetic modification may be associated with a coding region or a functional fragment thereof of a heterologous, a homologous, or a heterologous and homologous polypeptide with respect to the referenced species. In addition, the genetic modification may include modification in non-coding regulatory regions to modify expression of a gene or an operon, wherein the non-coding regulatory regions may include a 5′-non coding sequence and/or a 3′-non coding sequence.
The disruption of the gene may occur by genetic engineering methods, such as homologous recombination, directed mutagenesis, or molecular evolution. When a cell includes a plurality of the same genes or two or more paralogs of the gene, one or more genes may be disrupted. For example, the genetic modification may involve transformation in cells using a vector that includes a partial sequence of the gene, followed by the culturing of the cells in a cell culture medium, whereby homologous recombination of the partial sequence occurs with endogenous genes in the cells, so that the genes are disrupted. The cells that have undergone the homologous recombination are selected by using a selection marker, such as an antibiotic resistant gene.
The term “sequence identity” of a polypeptide or polynucleotide as used herein refers to a degree of sameness in an amino acid residue or a base in a specific region of two sequences that are aligned to best match each other for comparison. The sequence identity is a value obtained via alignment and comparison of the two sequences in the specific region for comparison, in which a partial sequence in the specific region for comparison may be added or deleted with respect to a reference sequence. The sequence identity represented in a percentage may be calculated by, for example, comparing two sequences that are aligned to best match each other in the specific region for comparison, determining matched sites with the same amino acid or base in the two sequences to obtain the number of the matched sites, dividing the number of the matched sites in the two sequences by a total number of sites in the compared specific regions (i.e., a size of the compared region), and multiplying a result of the division by 100 to obtain a sequence identity as a percentage. The sequence identity as a percentage may be determined using a known sequence comparison program, for example, BLASTP or BLASTN (NCBI), CLC Main Workbench (CLC bio), or MegAlignTM (DNASTAR Inc).
In identifying a polypeptide or polynucleotide with the same or similar function or activity with respect to various types of species, any various levels of sequence identity may be applied. For example, the sequence identity may be about 50% or more, about 55% or more, about 60% or more, about 65% 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, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or 100%.
The term “exogenous” as used herein denotes introduction of a referenced molecule (e.g., a polynucleotide) or a referenced activity to a host cell. The introduction of a molecule denotes, for example, introduction to a host genetic material of a nucleic acid encoding a protein or an enzyme by insertion of the exogenous nucleic acid into the host chromosomes, or introduction of the nucleic acid into a host cell as a non-chromosomal genetic material such as a plasmid. Regarding the expression of the encoding nucleic acid, the term “exogenous” denotes introduction of the encoding nucleic acid in an expressible form into a cell. In regard to the biosynthesis activity, the term “exogenous” denotes activity introduced to a host parent cell. The source of the exogenous gene may include, for example, homologous or heterologous nucleic acid that encodes a protein or an enzyme that expresses the referenced activity after being introduced to the host parent cell. Thus, the term “endogenous” denotes to a referenced molecule or a referenced activity that is already present in the host cell. Similarly, regarding the expression of the encoding nucleic acid, the term “endogenous” denotes expression of the nucleic acid encoding a protein or enzyme that is already expressed in the cell. The term “heterologous” denotes a molecule or an activity derived from a source other than the referenced species. The term “homologous” denotes a molecule or an activity from the host parent cell. Therefore, the exogenous expression of the nucleic acid encoding a protein or enzyme may include any one of heterologous or homologous encoding nucleic acids, or may include both heterologous and homologous nucleic acids encoding a protein or an enzyme.
The term “lactate” as used herein refers to lactic acid as well as its anion form, a salt thereof, a solvate, a polymorph, or a combination thereof. The salt may be, for example, an inorganic acid salt, an organic acid salt, or a metal salt. The inorganic acid salt may be hydrochloride, bromate, phosphate, sulfate, or disulfate. The organic acid salt may be formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edisylate, trifluoroacetate, benzoate, gluconate, methansulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, or aspartate. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.
According to one embodiment, there is provided a genetically engineered yeast cell with the capability of producing lactate and enhanced activity of an EDC protein as compared with activity of an EDC protein in a parent cell of the genetically engineered yeast cell.
The EDC protein may be an mRNA-decapping enhancer protein. The EDC may be an mRNA-binding protein that stimulates the mRNA decapping abilities of DCP1 and DCP2. The EDC may be EDC1 or EDC2. The EDC2 may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 1. For example, the EDC2 may have NCBI reference number of NP 010652.3. A gene encoding the EDC2 may be a polynucleotide having 95% or more sequence identity with respect to an amino acid sequence of SEQ ID NO: 2. The EDC1 may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 3. For example, the EDC1 may have NCBI reference number of NP_011293.1. A gene encoding the EDC1 may be a polynucleotide having 95% or more sequence identity with respect to an amino acid sequence of SEQ ID NO: 4.
The yeast cell may comprise modification in a regulatory sequence affecting gene expression, for example, a modification of the regulatory sequence of the gene encoding the EDC protein. The regulatory sequence affecting gene expression may include a sequence of a promoter for the gene expression or a sequence of a transcription terminator. The regulatory sequence affecting gene expression may include a sequence encoding a motif that influences the gene expression. The motif may include, for example, a secondary structure stability motif, an RNA instability motif, splice activation motif, a polyadenylation motif, an adenine-rich sequence, or an endonuclease recognition site. The sequence of the promoter may be an exogenous promoter that is operably linked to a gene encoding the EDC protein. The promoter may be a constitutive promoter. The promoter may be derived from covalently linked cell wall protein 12 (CCW12), pyruvate deCarboxylase 1 (PDC1), phosphoglycerate kinase (PGK1), transcription enhancer factor-1 (TEF-1), glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, or TDH3), triose phosphate isomerase (TPI1), purine-cytosine permease (PCPL3), or alcohol dehydrogenase (ADH1) genes. In addition, the regulatory sequence in gene expression may include a sequence that improves efficiency of translation. The sequence that improves efficiency of translation may be, for example, a sequence that improves initiation of the translation process, such as a Kozak consensus sequence.
The yeast cell may also have an increase in a copy number of the EDC protein-encoding gene. The yeast cell may include an exogenous gene that encodes the EDC protein. The exogenous gene may be appropriately regulated by an exogenous promoter that is operably linked thereto. The promoter is defined the same as described above.
The yeast cell may belong to Saccharomyces genus, Kluyveromyces genus, Candida genus, Pichia genus, Issatchenkia genus, Debaryomyces genus, Zygosaccharomyces genus, Shizosaccharomyces genus, or Saccharomycopsis genus. 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 yeast cell may have capability of producing L-lactate. The yeast cell may have an activity of a polypeptide that converts pyruvate to L-lactate. The yeast cell may include a gene encoding a polypeptide that converts pyruvate to L-lactate. In the yeast cell, the activity of a polypeptide that converts pyruvate to L-lactate may be increased. The polypeptide that converts pyruvate to L-lactate may be an enzyme that catalyzes the conversion of pyruvate to L-lactate, and for example, the polypeptide may be an L-lactate dehydrogenase (LDH). The LDH may be an NAD(P)-dependent enzyme. In addition, the LDH may be stereo-specific. The NAD(P)-dependent enzyme may be an enzyme that is classified under EC 1.1.1.27 that functions on L-lactate.
In the yeast cell capable of producing lactate, the activity of the LDH may be increased. The yeast cell may include a polynucleotide encoding at least one LDH, and the polynucleotide may be an exogenous gene. The polynucleotide encoding the LDH may be derived from bacteria, yeasts, fungi, mammals, or reptiles. The polynucleotide may be a polynucleotide that encodes at least one LDH selected from the group consisting of Lactobacillus helveticus, L. bulgaricus, L. johnsonii, L. plantarum, Pelodiscus sinensis japonicus, Ornithorhynchus anatinus, Tursiops truncatus, Rattus norvegicus, Xenopus laevis, and Bos taurus. An LDH derived from Bos taurus, an LDH derived from Pelodiscus sinensis japonicus, an LDH derived from Ornithorhynchus anatinus, an LDH derived from Tursiops truncatus, and an LDH derived from Rattus norvegicus may each have an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with amino acids of SEQ ID NOS: 13, 14, 15, 16, and 17. For example, a gene encoding the LDH may be a polynucleotide that encodes an amino acid sequence having 95% or more sequence identity with amino acid sequences of SEQ ID NOS: 13, 14, 15, 16, and 17. Alternatively, a gene encoding the LDH may have a polynucleotide sequence of SEQ ID NO: 18.
The gene encoding the LDH may be included in a vector. The vector may include a replication origin, a promoter, an LDH-encoding polynucleotide, 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 promoters of cytochrome c (CYC), transcription elongation factor (TEF), GPD, ADH, and CCW12 genes. The promoter of the CYC, TEF, GPD, ADH, and CCW12 genes may each include nucleotide sequences of SEQ ID NOS: 23, 24, 22, 25, and 21. The terminator may be selected from the group consisting of terminators of phosphoglycerate kinase 1 (PGK1), cytochrome c 1 (CYC1), galactokinase 1 (GAL1), and trehalose-6-phosphate synthase 1 (TPS1) genes. The terminator of the CYC1 gene may include a nucleotide sequence of SEQ ID NO: 26. The vector may further include a selection marker (e.g., an antibiotic selection marker). A polynucleotide encoding the LDH may be included in a genome at a specific site of the yeast cell. When the polynucleotide encoding the LDH functions on the production of active proteins in a cell, the polynucleotide is considered to be “functional” in a cell.
The yeast cell may include a polynucleotide that encodes one LDH or a polynucleotide that encodes multiple LDH copies, e.g., about 2 to about 10 copies. The yeast cell may include a polynucleotide that encodes multiple LDH copies into, for example, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 1 to about 4, or about 1 to about 3 copies. When the yeast cell includes the polynucleotide encoding multiple LDH copies, each polynucleotide may include copies of the same LDH or copies of at least two different LDHs. The multiple copies of the polynucleotide that encodes exogenous LDHs may be included in the same locus or multiple loci in a genome of a host cell, and the promoter or the terminator of each copy of the polynucleotide may be identical to or different from each other.
In the yeast cell, the activity of one or more pathways that interrupt the production or use of metabolic products involved in lactate production may be reduced. Additionally, the activity of one or more pathways that catalyze or assists in the production or use of metabolic products for producing lactate may be increased.
The yeast cell may additionally comprise a genetic modification such that the activity of a polypeptide that converts pyruvate to acetaldehyde, a polypeptide that converts lactate to pyruvate, a polypeptide that converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, a polypeptide that converts acetaldehyde to ethanol, aldehyde dehydrogenase, or a combination thereof may be reduced as compared with activity of one or more of these enzymes in a parent cell.
The yeast cell may include a disruption mutation of a gene encoding the polypeptide that converts pyruvate to acetaldehyde. The polypeptide that converts pyruvate to acetaldehyde may be an enzyme that catalyzes the conversion of pyruvate to acetaldehyde and is classified under EC 4.1.1.1. The polypeptide that converts pyruvate to acetaldehyde may be a pyruvate decarboxylase, and for example, may be PDC1. The polypeptide that converts pyruvate to acetaldehyde may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 5. The gene encoding the polypeptide that converts pyruvate to acetaldehyde may include a polynucleotide sequence encoding an amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NO: 5, or may include a polynucleotide sequence of SEQ ID NO: 6. For example, the gene may be PDC1.
The yeast cell may include a disruption mutation of a gene encoding the polypeptide that converts lactate to pyruvate. The polypeptide that converts lactate to pyruvate may be a CYC-dependent enzyme. The polypeptide that converts lactate to pyruvate may be an enzyme that is classified under EC 1.1.2.4 that acts on D-lactate, or that is classified under EC 1.1.2.3 that acts on L-lactate. The polypeptide that converts lactate into pyruvate may be lactate cytochrome c-oxidoreductase, for example, a CYB2 (CAA86721.1), a CYB2A, or a CYB2B. The polypeptide that converts lactate into pyruvate may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 7. The gene encoding the polypeptide that converts lactate into pyruvate may include a polynucleotide sequence encoding an amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NO: 7, or may include a polynucleotide sequence of SEQ ID NO: 8.
The yeast cell may include a disruption mutation of a gene encoding the polypeptide that converts DHAP to glycerol-3-phosphate. The polypeptide that converts DHAP to glycerol-3-phosphate may be a cytosolic glycerol-3-phosphatedehydrogenase, and may be an enzyme that catalyzes reduction of DHAP to glycerol-3-phosphate by using oxidation of NADH or NADP to NAD+ or NADP+. The polypeptide may be an enzyme that is classified under EC 1.1.1.8. The cytosolic glycerol-3-phosphate dehydrogenase may be GPD1. The cytosolic glycerol-3-phosphate dehydrogenase may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 9. The gene encoding the cytosolic glycerol-3-phosphate dehydrogenase may include a polynucleotide sequence encoding an amino acid sequence having a about 95% or more sequence identity with an amino acid sequence of SEQ ID NO: 9, or may include a polynucleotide sequence of SEQ ID NO: 10.
The yeast cell may include a disruption mutation of a gene encoding the polypeptide that converts pyruvate to D-lactate. The polypeptide may be classified under EC. 1.1.2.4. The polypeptide may be D-lactate dehydrogenase (DLD. The DLD may be also called D-lactate ferricytochrome C oxidoreductase. The polypeptide may be DLD1. The polypeptide may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 11. The gene encoding the polypeptide may include a polynucleotide sequence encoding an amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NO: 11. For example, the gene may include a polynucleotide of SEQ ID NO:
In the yeast cell, the yeast cell may include a disruption mutation of a gene encoding the polypeptide that converts acetaldehyde to ethanol. The polypeptide may be an enzyme that catalyzes the conversion of acetaldehyde to ethanol and that is classified under EC. 1.1.1.1. The polypeptide may be an enzyme that catalyzes the conversion of acetaldehyde to ethanol by involving conversion of NADH to NAD+. The polypeptide may be an alcohol dehydrogenase (Adh), and may be Adh1, Adh2, Adh3, Adh4, Adh5, or Adh6. The polypeptide may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 19. The gene encoding the polypeptide may include a polynucleotide sequence encoding an amino acid sequence having about 95% or more of sequence identity with an amino acid sequence of SEQ ID NO: 19, or may include a polynucleotide sequence of SEQ ID NO: 20. For example, the gene may be adh1, adh2, adh3, adh4, adh5, or adh6.
In addition, in the yeast cell according to an exemplary embodiment, the yeast cell may include increased activity of EDC as compared to a parent cell thereof; a disruption mutation of a gene encoding a polypeptide that converts pyruvate to acetaldehyde, a gene encoding a polypeptide that converts lactate to pyruvate, a gene encoding a polypeptide that converts DHAP to glycerol-3-phosphate, a gene encoding a polypeptide that converts pyruvate to D-lactate, a gene encoding a polypeptide that converts acetaldehyde to ethanol, or a combination thereof; and an exogenous gene encoding a polypeptide that converts pyruvate to L-lactate. The yeast cell may be a S. cerevisiae yeast cell.
According to another aspect, there is provided a composition for producing lactate, the composition including the yeast cell. The yeast cell is defined the same as described above.
According to another aspect, there is provided a method of producing a yeast cell that produces lactate, the method including: overexpressing a gene that encodes an EDC protein in a yeast cell; and introducing a gene encoding a polypeptide that converts pyruvate to L-lactate.
The method of producing the yeast cell that produces lactate may include a step of overexpressing a gene that encodes an EDC protein in the yeast cell. Here, the terms “yeast cell”, “EDC protein”, and “gene encoding the EDC protein” are defined the same as described above.
The step of overexpressing the gene encoding the EDC protein may result in overexpression of the EDC protein. That is, the overexpression of the EDC protein may denote that the yeast cell in which the gene encoding the EDC protein is overexpressed is produced in larger amounts or slightly higher steady-state as compared with the EDC protein in the parent cell so that the activity of the EDC protein is high in the yeast cell under the same conditions. In addition, the overexpression of the EDC protein may denote that mRNA encoding the EDC protein is produced in larger amounts or slightly higher steady-state as compared with the EDC protein in the parent cell so that the activity of the EDC protein is high in the yeast cell under the same conditions. Therefore, the overexpression of the EDC protein may be measured by using suitable enzyme analysis to measure an inactive level of the EDC protein in the host cell. The step of overexpressing the gene encoding the EDC protein may comprise introducing a genetic modification into the yeast cell that increases activity of the EDC protein, such as an increase in the copy number of the EDC protein, a modification of a regulatory sequence of a polynucleotide encoding an EDC protein, or a combination thereof.
The method of producing the yeast cell that produces lactate may include a step of introducing a gene encoding a polypeptide that converts pyruvate to L-lactate. In this step, the terms “polypeptide that converts to lactate” and “a gene encoding a polypeptide that converts to lactate” are defined the same as described above. The introduction of the gene may occur by using a vehicle such as a vector. The introduction may be transient introduction, in which the gene is not integrated into the genome, or may be integration of the gene into the genome. The introduction may occur by, for example, introducing a vector that is inserted with a polynucleotide encoding a desired polypeptide into the cell, and then, by replicating the vector in the cell or integrating the polynucleotide into the genome of the cell.
In addition, the method of producing the yeast cell that produces lactate may further include a step of disrupting a gene encoding a polypeptide that converts pyruvate to acetaldehyde, a gene encoding a polypeptide that converts lactate to pyruvate, a gene encoding a polypeptide that converts DHAP to glycerol-3-phosphate, a gene encoding a polypeptide that converts pyruvate to D-lactate, a gene encoding a polypeptide that converts acetaldehyde to ethanol, or a combination thereof. In this step, the terms “polypeptide that converts pyruvate to acetaldehyde”, “gene encoding the polypeptide that converts pyruvate to acetaldehyde”, “polypeptide that converts lactate to pyruvate”, “gene encoding the polypeptide that converts lactate to pyruvate”, “polypeptide that converts DHAP to glycerol-3-phosphate”, “gene encoding the polypeptide that converts DHAP to glycerol-3-phosphate”, “polypeptide that converts pyruvate to D-lactate”, “gene encoding the polypeptide converting pyruvate to D-lactate”, “polypeptide that converts acetaldehyde to ethanol”, “gene encoding the polypeptide converting acetaldehyde to ethanol”, and “disruption” are defined the same as described above.
According to another aspect, there is provided a method of producing lactate, the method including culturing the yeast cell in a cell culture medium, whereby the yeast cell produces lactate. The yeast cell is defined the same as described above.
The culturing of the yeast cell may be performed in a medium containing a carbon source, e.g., glucose. The medium used for the culturing of the yeast cell may be any conventional medium suitable for proliferation of the host cell, such as a minimal or complex medium containing appropriate supplements. The suitable medium may be available from a commercial seller or may be manufactured according to known manufacturing methods. The medium for the culturing of the yeast cell may be a medium that satisfies requirements of a particular yeast cell. The medium may be selected from media consisting of carbon sources, nitrogen sources, salts, trace elements, and a combination thereof.
The culture conditions may be appropriately controlled to obtain lactate from the genetically engineered yeast cell. The yeast cell may be cultured in aerotropic conditions. In order to produce lactate, the yeast cell may be cultured in microaerobic or anaerobic conditions. The term “anaerobic conditions” refers to an environment without oxygen. When the term “microaerobic conditions” is used by referring to culture or growth conditions, the microaerobic conditions may include maintaining a dissolved oxygen (DO) concentration of the medium in a range of about 0% to about 10% with respect to a DO concentration of a liquid medium in a saturation state. The microaerobic conditions also include growing or resting cells in a liquid medium or on a solid agar plate placed in a sealed chamber maintained in an atmosphere having oxygen of less than 1%. The concentration of oxygen may be maintained by, for example, sparging the culture in a mixture of N2/CO2 or in other suitable non-oxygen gases. The oxygen conditions may include a case where the DO concentration is maintained in a range of about 0% to about 10%, for example, about 0% to about 8%, about 0% to about 6%, about 0% to about 4%, or about 0% to about 2%.
The term “culture conditions” may denote conditions required for culturing the yeast cells. The culture conditions may include, for example, a carbon source or a nitrogen source, or oxygen conditions available for the yeast cell. The carbon source available for the yeast cell may include a monosaccharide, a disaccharide, or a polysaccharide, and examples thereof are glucose, fructose, mannose, or galactose. The nitrogen source available for the yeast cell may include an organic nitrogen compound or an inorganic nitrogen compound. Examples of the nitrogen source are amino acids, amides, amines, nitrates, or ammonium salts.
The method of producing lactate may further include collecting lactate from the culture.
The collecting of lactate from the culture may be carried out by using separation methods that are commonly known in the art. The separation methods may include centrifugation, filtration, ion-exchange chromatography, or crystallization. For example, the culture may be centrifuged at a low speed to remove biomass, and then, a supernatant obtained therefrom may be separated through ion-exchange chromatography.
The yeast cell according to an aspect may be used to produce lactate in an effective manner.
The method of producing the yeast cell according to an aspect may be used to produce the yeast cell producing lactate in an effective manner.
The method of producing lactate according to an aspect may be used to produce lactate in an effective manner.
Hereinafter, the present inventive concept 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: Manufacture of Strain with Capability of Producing L-Lactate
In order to prepare a S. cerevisiae CEN.PK2-1 D wild-type strain (MATaura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2, EUROSCARF accession number: 30000B) as a strain for producing lactate, gene deletion occurred in main enzymes involved in alcohol fermentation, i.e., a pyruvate decarboxylase (pdc1) gene and an alcohol dehydrogenase (adh1) gene, a main enzyme involved in biosynthesis of glycerol, i.e., a NAD-dependent glycerol-3-phosphate dehydrogenase (gpd1) gene, and proteolytic enzymes of lactate, i.e., an L-lactate cytochrome-c oxidoreductase 2 (cyb2) gene and a D-lactate dehydrogenase (dld1) gene. Then, a lactate dehydrogenase (BtLDH) gene derived from Bos Taurus was inserted at the adh1 gene site, thereby manufacturing an MD1304 strain for producing lactate. Each procedure of the strain manufacture is as follows.
1.1: Manufacture of Strain Including pdc1 Deletion
A 260 bp DNA fragment (hereinafter, referred to as ‘Fragment 1’) was obtained as an amplification product by PCR that was performed by using a genome DNA of the S. cerevisiae CEN.PK2-1D wild-type strain as a template and a primer set of SEQ ID NOS: 27 and 28 as primers. A 257 bp DNA fragment (hereinafter, referred to as ‘Fragment 2’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 29 and 30 as primers. A 1,955 bp DNA fragment (hereinafter, referred to as ‘Fragment 3’) was obtained as an amplification product by PCR that was performed by using a pCtB1 vector (GenBank Accession Number KJ922019) as a template and a primer set of a T7 primer and a T3 primer as primers. FIG. 1 is a view of a pCtB1 vector (GenBank Accession Number KJ922019). Here, the pCtB1 vector had a polynucleotide of SEQ ID NO: 67. These DNA fragments, i.e., Fragments 1, 2, and 3, were combined together, and then, subjected to PCR using a primer set of SEQ ID NOS: 27 and 30 as primers, so as to obtain a 2,414 bp DNA fragment, which is a DNA fragment used to substitute a URA3 terminator BLAST for the PDC1 gene.
Transformation of the DNA fragment occurred in the S. cerevisiae CEN.PK2-1 D wild-type strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among the selected strains, a strain having identification of a 2,947 bp DNA fragment was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 31 and 32, wherein the primer set had identification of a 2,904 bp DNA fragment from the wild-type strain.
The obtained strain was subjected to a seed culture for one day in an YPD culture medium (Yeast Extract 10 g, Bacto Peptone 20 g, D-glucose 20 g/L). Afterwards, colonies were spread on a 5-FOA culture medium (Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, uracil 0.1 g, D-glucose 20 g, 5-fluoroorotic acid 1 g, Bacto Agar 20 g/L) to select strains that were grown and developed therefrom. Among the selected strains, PCR was performed again by using a primer set of SEQ ID NOS: 31 and 32, so as to obtain a strain having identification of a 1,553 bp DNA fragment, and that is, a S. cerevisiae CEN.PK2-1D (Δpdc1) strain (hereinafter, referred to as ‘MD1002 strain’) was obtained.
1.2: Manufacture of Strain Including gpd1 Deletion
A 278 bp DNA fragment (hereinafter, referred to as ‘Fragment 4’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 33 and 34 as primers. A 446 bp DNA fragment (hereinafter, referred to as ‘Fragment 5’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 35 and 36 as primers. A 1,955 bp DNA fragment (hereinafter, referred to as ‘Fragment 6’) was obtained as an amplification product by PCR that was performed by using a pCtB1 vector (GenBank Accession Number KJ922019) as a template and a primer set of a T7 primer and a T3 primer as primers. These DNA fragments, i.e., Fragments 4, 5, and 6, were combined together, and then, subjected to PCR using a primer set of SEQ ID NOS: 33 and 36 as primers, so as to obtain a 2,621 bp DNA fragment, which is a DNA fragment used to substitute a URA3 terminator BLAST for the GPD1 gene.
Transformation of the DNA fragment occurred in the MD1002 strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among the selected strains, a strain having identification of a 2,860 bp DNA fragment was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 37 and 38, wherein the primer set had identification of a 2,160 bp DNA fragment in the wild-type strain or the MD1002 strain. The obtained strain was subjected to a seed culture for one day in an YPD culture medium YPD (Yeast Extract 10 g, Bacto Peptone 20 g, D-glucose 20 g/L). Afterwards, colonies were spread on a 5-FOA culture medium (Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, uracil 0.1 g, D-glucose 20 g, 5-fluoroorotic acid 1 g, Bacto Agar 20 g/L) to select strains that were grown and developed therefrom. Among the selected strains, PCR was performed again by using a primer set of SEQ ID NOS: 37 and 38, so as to obtain a strain having identification of a 1,466 bp DNA, and that is, a S. cerevisiae CEN.PK2-1 D stain (Δpdc1 Δgpd1) (hereinafter, referred to as ‘MD1228 strain’) was obtained.
1.3: Manufacture of Strain Including cyb2 Deletion
A 355 bp DNA (hereinafter, referred to as ‘Fragment 7’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 39 and 40 as primers. A 378 bp DNA fragment (hereinafter, referred to as ‘Fragment 8’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1D wild-type strain as a template and a primer set of SEQ ID NOS: 41 and 42 as primers. A 1,955 bp DNA fragment (hereinafter, referred to as ‘Fragment 9’) was obtained as an amplification product by PCR that was performed by using a pCtB1 vector (GenBank Accession Number KJ922019) as a template and a primer set of a T7 primer and a T3 primer as primers. These DNA fragments, i.e., Fragments 7, 8, and 9, were combined together, and then, subjected to PCR using a primer set of SEQ ID NOS: 39 and 42, so as to obtain a 2,630 bp DNA fragment, which is a DNA fragment used to substitute a URA3 terminator BLAST for the CYB2 gene.
Transformation of the DNA fragment occurred in the MD1128 strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among the selected strains, a strain having identification of a 2,891 bp DNA was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 43 and 44, wherein the primer set had identification of a 2,676 bp DNA in the MD1002 strain or the MD1228 strain. The obtained strain was subjected to a seed culture for one day in an YPD culture medium YPD (Yeast Extract 10 g, Bacto Peptone 20 g, D-glucose 20 g/L). Afterwards, colonies were spread on a 5-FOA culture medium (Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, uracil 0.1 g, D-glucose 20 g, 5-fluoroorotic acid 1 g, Bacto Agar 20 g/L) to select strains that were grown and developed therefrom. Among the selected strains, PCR was performed again by using a primer set of SEQ ID NOS: 43 and 44, so as to obtain a strain having identification of a 1,497 bp DNA, and that is, a S. cerevisiae CEN.PK2-1 D stain (Δpdc1, Δgpd1, Δcyb2) (hereinafter, referred to as ‘MD1258 strain’) was obtained.
1.4: Manufacture of Strain Including dld1 Deletion
A 366 bp DNA (hereinafter, referred to as ‘Fragment 10’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 45 and 46 as primers. A 365 bp DNA fragment (hereinafter, referred to as ‘Fragment 11’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1D wild-type strain as a template and a primer set of SEQ ID NOS: 47 and 48 as primers. A 1,955 bp DNA fragment (hereinafter, referred to as ‘Fragment 12’) was obtained as an amplification product by PCR that was performed by using a pCtB1 vector (GenBank Accession Number KJ922019) as a template and a primer set of a T7 primer and a T3 primer as primers. These DNA fragments, i.e., Fragments 10, 11, and 12, were combined together, and then, subjected to PCR using a primer set of SEQ ID NOS: 45 and 48, so as to obtain a 2,628 bp DNA fragment, which is a DNA fragment used to substitute a URA3 terminator BLAST for the DLD1 gene.
Transformation of the DNA fragment occurred in the MD1258 strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among the selected strains, a strain having identification of a 2,891 bp DNA was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 49 and 50, wherein the primer set had identification of a 3,209 bp DNA in the wild-type strain, the MD1002 strain, the MD1228 strain, or the MD1258 strain. The obtained strain was subjected to a seed culture for one day in an YPD culture medium YPD (Yeast Extract 10 g, Bacto Peptone 20 g, D-glucose 20 g/L). Afterwards, colonies were spread on a 5-FOA culture medium (Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, uracil 0.1 g, D-glucose 20 g, 5-fluoroorotic acid 1 g, Bacto Agar 20 g/L) to select strains that were grown and developed therefrom. Among the selected strains, PCR was performed again by using a primer set of SEQ ID NOS: 49 and 50, so as to obtain a strain having identification of a 41,815 bp DNA fragment, and that is, a S. cerevisiae CEN.PK2-1D strain (Δpdc1, Δgpd1, Δcyb2, Δdld1) (hereinafter, referred to as ‘MD1257 strain’) was obtained.
1.5: Manufacture of Vector for LDH Overexpression
A 1,605 bp DNA fragment including a CCW12 promoter was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 51 and 52 as primers. The DNA fragment was combined with a pCtB1 vector treated with KpnI, and cloned by using an In-fusion kit, so as to obtain a pCtB1-CCW12 vector (hereinafter, referred to as ‘MD0922 vector’), which is a dual-purpose expression vector. The MD0922 vector was treated with KpnI and SacI, combined with a DNA fragment of the LDH gene derived that was derived from Bos Taurus (hereinafter, referred to as ‘BtLDH’) and synthesized as SEQ ID NO: 18, and cloned to a pCS-Ex1.1 vector by using an In-fusion kit, so as to obtain a pCS-Ex1.1 BtLDH vector (hereinafter, referred to as ‘MD1188’ vector), which is a dual-purpose vector for the overexpression of the LDH gene in yeast cell. The BtLDH gene was set to involve transcription in the presence of the CCW12 promoter. FIG. 2 is a view of a pCS-Ex1.1 vector.
1.6: Manufacture of Strain Including Adh1 Deletion and Having Capability of Producing Lactate
A 333 bp DNA fragment (hereinafter, referred to as ‘Fragment 13’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1D wild-type strain as a template and a primer set of SEQ ID NOS: 53 and 54 as primers. A 397 bp DNA fragment (hereinafter, referred to as ‘Fragment 14’) was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 55 and 56 as primers. A 4,422 bp DNA fragment (hereinafter, referred to as ‘Fragment 15’) was obtained as an amplification product by PCR that was performed by using the MD1188 vector as a template and a primer set of a T7 primer and a T3 primer as primers. These DNA fragments, i.e., Fragments 12, 14, and 15, were combined together, and then, subjected to PCR using a primer set of SEQ ID NOS: 53 and 56, so as to obtain a 5,094 bp DNA fragment, which is a DNA fragment used to substitute a CCW12 promoter-BtLDH gene-TPS1 terminator-URA3 terminator BLAST for the ADH1 gene.
Transformation of the DNA fragment occurred in the MD1257 strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among the selected strains, a strain having identification of a 5276 bp DNA was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 57 and 58, wherein the primer set had identification of a 1,918 bp DNA in the wild-type strain, the MD1002 strain, the MD1228 strain, or the MD1258 strain. The obtained strain was subjected to a seed culture for one day in an YPD culture medium YPD (Yeast Extract 10 g, Bacto Peptone 20 g, D-glucose 20 g/L). Afterwards, colonies were spread on a 5-FOA culture medium (Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, uracil 0.1 g, D-glucose 20 g, 5-fluoroorotic acid 1 g, Bacto Agar 20 g IL) to select strains that were grown and developed therefrom. Among the selected strains, PCR was performed again by using a primer set of SEQ ID NOS: 57 and 58, so as to obtain a strain having identification of a 3,882 bp DNA fragment, and that is, a S. cerevisiae CEN.PK2-1D strain (Δpdc1, Δgpd1, Δcyb2, Δdld1, Δadh1::Btldh) (hereinafter, referred to as ‘MD1304 strain’) was obtained.
Example 2: Manufacture of Strain for Overexpression of EDC2
2.1: Manufacture of Vector for Overexpression of EDC2
In order to overexpress the EDC2 gene in the MD1304 strain having capability of producing lactate, a vector for overexpression of EDC2 was manufactured. Here, a 479 bp DNA fragment including the EDC2 gene was obtained as an amplification product by PCR that was performed by using the genome DNA of the S. cerevisiae CEN.PK2-1 D wild-type strain as a template and a primer set of SEQ ID NOS: 59 and 60 as primers. The DNA fragment was combined with a DNA of a pRS426GPD vector treated with XbaI and XhoI to carry out cloning in the pRS426GPD vector by using an In-fusion kit, so as to obtain a pRS426GPD EDC2 vector as a vector for overexpression of EDC2. Afterwards, a 1,168 bp DNA fragment was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 61 and 62 as primers from the pRS426GPD EDC2 vector. The DNA fragment was used to clone the GPD promoter-EDC2 gene to pCtB1. The DNA fragment was combined with DNA of the pCtB1 vector treated with KpnI and SacI, and then, cloned to the pCS-Ex1 by using an In-fusion kit, so as to obtain a MD1152 vector (i.e., pCS-Ex1 EDC2 vector), which is a dual-purpose vector for overexpression of EDC2. FIG. 3 is a view of a pCS-Ex1 vector. Here, the EDC2 gene was set to involve transcription in the presence of the GPD promoter.
2.2: Manufacture of Strain for Overexpression of EDC2 Overexpression and Manufacture of Control Strain
In order to overexpress the EDC2 gene in the MD1304 strain having capability of producing lactate, an EDC2 overexpression gene cassette was inserted by being substituted with a PDC6 gene having no known gene function. A 3,057 bp DNA was obtained as an amplification product by PCR that was performed by using a MD1152 vector, which is a dual-purpose vector for overexpression of EDC2, as a template and a primer set of SEQ ID NOS: 63 and 64 as primers. That is, the DNA fragment was used to substitute the PDC6 for GPD promoter-EDC2-TPS1 terminator-URA3 terminator BLAST.
Transformation of the DNA fragment occurred in the S. cerevisiae CEN.PK2-1 D wild-type strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among selected strains, a strain having identification of a 3,429 bp DNA fragment was obtained by PCR that was performed by using a primer set of SEQ ID NOS: 65 and 66, wherein the primer set had identification of a 2,300 bp DNA fragment in the wild-type strain, and that is, a S. cerevisiae CEN.PK2-1D stain (Δpdc1, Δgpd1, Δcyb2, Δdld1, Δadh1::Btldh, Δpdc6::EDC2) (hereinafter, referred to as ‘MD1304/EDC2 strain’) was obtained.
In order to manufacture a control strain, a 2,029 bp DNA fragment was obtained as an amplification product by PCR that was performed by using an empty pCtB1 vector as a template and a primer set of SEQ ID NOS: 63 and 64 as primers. Transformation of the DNA fragment occurred in the S. cerevisiae CEN.PK2-1D wild-type strain, and then, strains grown on a minimal medium without containing uracil (SD-URA; Yeast Nitrogen Base 6.7 g, Synthetic Drop-out without uracil 1.92 g, D-glucose 20 g, Bacto Agar 20 g/L) were selected therefrom. Among the selected strains, a strain having identification of a 2,301 bp DNA fragment was obtained from the wild-type strain by PCR that was performed by using a primer set of SEQ ID NOS: 65 and 66, wherein the primer set had identification of a 2,300 bp DNA fragment in the wild-type strain, and that is, a S. cerevisiae CEN.PK2-1D strain (Δpdc1, Δgpd1, Δcyb2, Δdld1, Δadh1::Btldh, Δpdc6) (hereinafter, referred to as ‘MD1304/Δpdc6 strain’) was obtained.
Example 3: Evaluation of Capability of Producing Lactate of Strain for Overexpression of EDC2
In order to evaluate effects of the overexpression of the EDC2 gene on a strain's capability of producing lactate, a typical batch flask test was carried out. Here, the MD1304/Δpdc6 strain and the MD1304/EDC2 strain under the exponential-growth phase were collected and inoculated into a YPD8 culture medium (Yeast Extract 10 g, Bacto Peptone 20 g, D-Glucose 80 g/1 L), which is a test medium for the batch flask test, thereby achieving optical density (OD) of 4.0 in the cell concentration of 600 nm. The cell-medium mixture was put in an incubator under microaerobic conditions including humidity of 95% or above and maintaining oxygen concentration of 2.5%, and then, the cell-medium mixture was sufficiently stirred and cultured at a temperature of 30° C.
As shown in Table 1 below, the results show that the MD1304/EDC2 strain where the EDC2 was overexpressed had better capability of producing lactate and greater lactate yields than those of the control group, i.e., the MD1304/Δpdc6 strain.












TABLE 1





Strain

0 hour
20 hours
40 hours
71 hours







MD1304/Δ pdc 6
OD600
3.97 ± 0.01
7.85 ± 0.27
10.96 ± 0.25
11.64 ± 0.18



D-glucose
84.8 ± 2.7
52.9 ± 1.2 
 40.5 ± 1.6
 38.9 ± 0.8



(g/L)


 
 



L-lactate
 0.2 ± 0.1
17.7 ± 0.5 
 25.2 ± 0.6
 25.5 ± 0.7



(g/L)


 
 



Yield (g/g %)
—
  55 ± 3.7
 56.6 ± 3.0
 55.2 ± 1.4


MD1304/EDC2
OD600
4.04 ± 0.10
6.89 ± 0.23
 9.17 ± 0.98 
10.91 ± 0.48



D-glucose
84.8 ± 2.7
50.8 ± 1.5
 35.9 ± 1.6
 25.2 ± 0.3



(g/L)


 
 



L-lactate
 0.2 ± 0.1
21.3 ± 0.3
 31.2 ± 0.9
 35.4 ± 0.2



(g/L)


 
 



Yield (g/g %)
—
62.1 ± 2.0
 63.5 ± 3.1
 59.1 ± 0.6


Δ (EDC2)
OD600
—
−12.2%
−16.3%
 −6.3%



D-glucose
—
 +6.6%
+10.6%
+29.7%



(g/L)







L-lactate
—
+20.4 %
+24.1%
+38.8%



(g/L)







Yield (g/g %)
—
+12.9%
+12.2%
 +7.1%





* Average ± S.D (n = 3)






In Table 1, the measured OD600, D-glucose, L-lactate, and yields in the Δ (EDC2) showed increased values of the MD1304/EDC2 associated with increased activity of the EDC2, as compared with those of the MD1304/Δ pdc6.
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 exemplary embodiment should typically be considered as available for other similar features or aspects in other exemplary embodiments.
While one or more exemplary embodiments 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 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 that produces lactate, wherein the genetically engineered yeast cell comprises a genetic modification that increases copy number of a polynucleotide encoding an enhancer of mRNA-decapping (EDC) protein as compared to the same yeast cell without the genetic modification that increases copy number, wherein the genetically modified yeast cell comprises an exogenous polynucleotide encoding a lactate dehydrogenase, and wherein the genetically modified yeast cell comprises a disruption mutation of a chromosomal gene encoding a polypeptide that converts pyruvate to acetaldehyde.

2. The genetically engineered yeast cell of claim 1, wherein the genetic modification that increases copy number is an exogenous promoter that is operably linked to the polynucleotide encoding the EDC protein.

3. The genetically engineered yeast cell of claim 1, wherein the genetic modification that increases copy number is an exogenous polynucleotide encoding the EDC protein.

4. The genetically engineered yeast cell of claim 1, wherein the EDC protein is an EDC1 protein or an EDC2 protein.

5. The genetically engineered yeast cell of claim 4, wherein the EDC2 protein comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and has mRNA-decapping enhancer activity.

6. The genetically engineered yeast cell of claim 4, wherein the EDC2 protein comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and has mRNA-decapping enhancer activity.

7. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell belongs to the Saccharomyces genus, the Kluyveromyces genus, the Candida genus, the Pichia genus, the Issatchenkia genus, the Debaryomyces genus, the Zygosaccharomyces genus, the Shizosaccharomyces genus, or the Saccharomycopsis genus.

8. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell is a Saccharomyces cerevisiae yeast cell.

9. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell produces lactate under anaerobic, aerobic or microaerobic conditions.

10. The genetically engineered yeast cell of claim 1, wherein the lactate dehydrogenase comprises an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 13, 14, 15, 16 or 17 and has lactate dehydrogenase activity.

11. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell further comprises a disruption mutation of a chromosomal gene encoding a polypeptide that converts lactate to pyruvate, a chromosomal gene encoding a polypeptide that converts dehydroxy acetone phosphate (DHAP) to glycerol-3-phosphate, a chromosomal gene encoding a polypeptide that converts pyruvate to D-lactate, a chromosomal gene encoding a polypeptide that converts acetaldehyde to ethanol, or a combination thereof.

12. A method of producing the genetically engineered yeast cell of claim 1, the method comprising:
transforming a yeast cell with a polynucleotide that encodes an EDC protein,
transforming the yeast cell with a polynucleotide that encodes a lactate dehydrogenase, and
disrupting a chromosomal gene encoding a polypeptide that converts pyruvate to acetaldehyde in the yeast cell,
thereby producing the genetically engineered yeast cell.

13. The method of claim 12, further comprising disrupting a chromosomal gene encoding a polypeptide that converts lactate to pyruvate, a chromosomal gene encoding a polypeptide that converts dehydroxy acetone phosphate (DHAP) to glycerol-3-phosphate, a chromosomal gene encoding a polypeptide that converts pyruvate to D-lactate, a chromosomal gene encoding a polypeptide that converts acetaldehyde to ethanol, or a combination thereof.

14. The method of claim 12, wherein the yeast cell is a Saccharomyces cerevisiae yeast cell.

15. A method of producing lactate, the method comprising:
culturing the genetically engineered yeast cell of claim 1 in a cell culture medium, whereby the genetically engineered yeast cell produces lactate.

16. The method of claim 15, further comprising collecting the lactate from the cell culture medium.

17. The method of claim 15, wherein the culturing of the genetically engineered yeast cell is performed under a microaerobic condition.