# Patent

## Title
Yeast cell having decreased RGT1 activity, method of producing the same, and method of producing product using the same

## Abstract

A recombinant yeast cell having a decreased RGT1 protein activity and an increased ability to produce a glycolytic intermediate or a glycolytic intermediate-derived substance, compared to a parent cell; methods of producing the same; and methods of producing the glycolytic intermediate or the glycolytic intermediate-derived substance using the same.

## Background
RELATED APPLICATION
This application claims the benefit of Korean Patent Application No. 10-2014-0191130, filed on Dec. 26, 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 145,131 Byte ASCII (Text) file named, “720522_ST25.TXT-Revised” created on Mar. 17, 2016.
BACKGROUND
1. Field
The present disclosure relates to a recombinant (i.e., genetically engineered) yeast cell that produces a glycolytic intermediate or a glycolytic intermediate-derived substance, a method of producing the same, and a method of producing the glycolytic intermediate or the glycolytic intermediate-derived substance using the same.
2. Description of the Related Art
Products such as organic acids and alcohols have been widely used as building block materials in the food, medical, and chemical industries. These substances have been known to be produced from petroleum, but methods of producing the substances using environmentally friendly microorganisms, such as yeasts, have also been studied.
These methods of producing products using microorganisms require a long fermentation time and high cost for product isolation. Thus there is a demand to improve the productivity of the microorganisms in the methods of producing products such as organic acids and alcohols.
Many approaches to increase productivity rely on the assumption that a production environment characteristic, such as acid stress, limits the productivity of microorganisms in said production environment. A main focus for strain improvement relates to the product formation rate itself by enhancing the enzymatic activities of microorganism that are involved in product formation. Examples of the enzymatic activities, which are generally enhanced, may include central metabolic pathways such as glycolysis, which provide intermediates required for the production of products.
Accordingly, there is still demand for a yeast cell having an enhanced ability to produce a product such as an organic acid or alcohol, as well as methods of producing the product using the same.
SUMMARY
A genetically engineered yeast cell having a decreased RGT1 protein activity compared to the RGT1 protein activity of a parent cell thereof, wherein the genetically engineered yeast cell has an increased productivity of a glycolytic intermediate or a glycolytic intermediate-derived substance compared to a parent cell thereof, and the genetically engineered yeast cell comprises a genetic modification that decreases the RGT1 protein activity. Another aspect provides a method of producing the genetically engineered yeast cell capable of effectively producing a glycolytic intermediate or the glycolytic intermediate-derived substance comprising introducing a gene encoding an enzyme that catalyzes the conversion of pyruvate into lactate into a yeast cell; and disrupting a gene encoding an RGT1 protein in the yeast cell.

Still another aspect provides a method of effectively producing the glycolytic intermediate or the glycolytic intermediate-derived substance using the genetically engineered yeast cell introducing a gene encoding an enzyme that catalyzes the conversion of pyruvate into lactate into a yeast cell; and disrupting a gene encoding an RGT1 protein in the yeast cell.




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 shows a cleavage map of a pJ1214-MhpF vector;
FIG. 2 shows a cleavage map of a p416-CCW12-LDH_HPH vector; and
FIG. 3 shows a cleavage map of a pUC19_Trp1_Pcyc1 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 merely described below, by referring to the figures, to explain aspects.
As used herein, the term “decrease in activity” or “decreased activity” means that a cell (e.g., a genetically engineered yeast cell) has an activity of an enzyme or a polypeptide that is lower than that measured in a parent cell. Also, the “decrease in activity” or “decreased activity” may refer to situations where an isolated enzyme or a polypeptide has an activity that is lower than that of an original or a wild-type enzyme or polypeptide (e.g., an enzyme or polypeptide from a parent cell). The decrease in activity or decreased activity encompasses no activity. For example, a modified (e.g., genetically engineered) cell or enzyme may have an enzymatic activity of converting a substrate to a product, which is 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% decreased, compared to that of a cell or enzyme that does not have the genetic modification, e.g., an enzyme from a parent cell or a “wild-type” cell or enzyme. Decreased activity of an enzyme or a cell may be confirmed by any method known in the art, for example, the activity of an enzyme may be measured by a colorimetric assay or a radiometric assay. The decrease in activity of an enzyme includes situations in which an enzyme has no activity or has decreased activity even though the enzyme is expressed, or the case that an enzyme-encoding gene is not expressed or is expressed at a low level, compared to a cell having a non-modified gene, i.e., a parent cell or a wild-type cell. The cell having decreased activity may have one or more genetic modifications for decreasing the activity of the enzyme or polypeptide, compared to a cell having no genetic modification.
With respect to a particular genetic modification, the “parent cell” may be a cell that lacks a particular genetic modification or set of modifications, but is identical in all other respects. A parent cell, thus, includes but is not limited to a non-genetically engineered cell of the same type as an engineered yeast cell (e.g., a “wild-type” cell) or a genetically engineered cell that serves as a starting point for further modifications that results in a particular genetically engineered cell. Thus, the parent cell may be a cell that is used as a starting material to produce a genetically engineered yeast cell having decreased or increased biological activity, such as the activity of a given protein or enzyme (e.g., a protein having a sequence identity of about 95% or more to an RGT1 protein or a protein having a sequence identity of about 95% or more to an HXK2 protein).
As used herein, the term “disruption” and “disruption mutation” refers to a genetic modification to reduce the expression of a referenced gene. The disruption includes a genetic manipulation whereby the referenced gene is not expressed (hereinafter, referred to as “inactivation” of a gene) or a genetic manipulation whereby the gene is expressed at a reduced level (hereinafter, referred to as “attenuation” of a gene). The inactivation includes not only the lack of expression of any product of a gene but also expression of a non-functional product even though the gene is expressed. Attenuation includes a reduction in the expression level of a functional product of a gene. That is, the attenuation includes situations where there is a reduction in the expression level of the functional product even if the entire expression of the gene is increased. Herein, the functional product of the gene refers to a product retaining a biochemical or physiological function (e.g., enzymatic activity) of the product (e.g., enzyme) encoded by the gene of a parent cell or a wild-type cell. As used herein the term “disruption” also includes functional disruption of the gene caused by a genetic modification. Genetic modifications which may result in the disruption of a gene include introducing a polynucleotide encoding a polypeptide into a cell; a substitution, insertion, or deletion of one or more nucleotides in the genetic material of a parent cell; or a chemical modification of the genetic material of the parent cell. Such genetic modifications include modification of coding regions and functional fragments thereof. In addition, the genetic modifications include modifications of non-coding regulatory regions, which alter the expression of a gene or an operon. The non-coding regions include a 5′-non coding sequence and/or a 3′-non coding sequence.
The disruption of a gene may be achieved by genetic manipulation such as homologous recombination, directed mutagenesis, or molecular evolution. If a cell includes a plurality of the same genes, or two or more different paralogs of a gene, one or more of the genes may be disrupted. For example, the genetic modification may be performed by transforming the cell with a vector containing a partial sequence of the gene, culturing the cell so that the gene is disrupted by homogonous recombination of the sequence with an endogenous gene of the cell, and then selecting cells, in which the homologous recombination occurred, using a selection marker.
The term “increase in activity” or “increased activity”, as used herein, may refer to a detectable increase in an activity of a cell, a protein, or an enzyme. The “increased activity” or “increase in activity” may also refer to an activity level of a modified (e.g., genetically engineered) cell, protein, or enzyme that is higher than that of a comparative cell, protein, or enzyme of the same type, such as a cell, protein, or enzyme that does not have a given modification (e.g., parent cell or “wild-type” cell, protein, or enzyme). “Cell activity” may refer to an activity of a particular protein or enzyme of a cell. For example, an activity of a modified or engineered cell, protein, or enzyme 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 than an activity of a cell, protein, or enzyme of the same type that does not have the genetic modification, e.g., a parent cell or protein or enzyme from a parent cell. A cell having an increased activity of a protein or an enzyme may be identified by using any method known in the art. For instance, the increased activity of a protein or an enzyme may be identified by measuring an amount of a product which is prepared from the reaction catalyzed by the protein or the enzyme. The cell having the increased activity may have one or more genetic modifications of increasing the activity of the enzyme or polypeptide, compared to a cell without the genetic modifications.
An increased activity of a polypeptide may be achieved by increasing an expression or a specific activity of the polypeptide. The increased expression may be caused by introducing a polynucleotide encoding the polypeptide into a cell, by increasing the copy number of an existing gene in the cell by amplification, or by modifying a regulatory region of the polynucleotide. Increasing the gene copy number may be achieved, for instance, by introduction of a polynucleotide encoding a gene, change of a promoter from a relatively weak promoter to a relatively strong promoter, or selective pressures advantageous to gene expression. The modification in the regulatory region of the polynucleotide may have a modification in an expression regulatory sequence of the gene. The regulatory sequence may be a promoter sequence or a transcription terminator sequence for expression of the gene. The regulatory sequence may also be a nucleotide sequence encoding a motif which may influence the expression of the gene. The motif may be, for example, a secondary structure-stabilizing motif, an RNA destabilizing motif, a splice-activating motif, a polyadenylation motif, an adenine-rich sequence, or an endonuclease recognition site.
The “increase in the copy number” may be caused by introduction or amplification of a gene, and may be achieved by genetically engineering a cell so that the cell possesses a gene that does not exist in a non-engineered cell or parent cell. The introduction of the gene may be mediated by a vehicle such as a vector. The introduction may be a transient introduction in which the gene is not integrated into a genome, or by non-transient integration of the gene into the genome. The introduction may be performed, for example, by introducing a vector into the cell, in which the vector includes a polynucleotide encoding a target polypeptide, and then, replicating the vector in the cell, or by integrating the polynucleotide into the genome.
As used herein, the term “gene” refers to a nucleic acid fragment expressing a specific protein, and may or may not include regulatory elements such as a 5′-non coding sequence and/or a 3′-non coding sequence.
As used herein, the term “sequence identity” of a nucleic acid or a polypeptide refers to a degree of identity between bases or amino acid residues of sequences obtained after the sequences are aligned so as to best match in certain comparable regions. The sequence identity is a value that is measured by comparing two sequences in certain comparable regions via optimal alignment of the two sequences, in which portions of the sequences in the certain comparable regions may be added or deleted compared to reference sequences. A percentage of sequence identity may be calculated by, for example, comparing two optimally aligned sequences in the entire comparable regions, determining the number of locations in which the same amino acids or nucleic acids appear to obtain the number of matching locations, dividing the number of matching locations by the total number of locations in the comparable regions (that is, the size of a range), and multiplying a result of the division by 100 to obtain the percentage of the sequence identity. The percentage of the sequence identity may be determined using a known sequence comparison program, for example, BLASTN and BLASTP (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc.
Various levels of sequence identity may be used to identify various types of polypeptides or polynucleotides having the same or similar functions or activities. For example, a sequence identity of 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% may be used as a reference.
As used herein, the term “exogenous” means that a referenced molecule or a referenced activity is introduced into a host cell. The molecule can be introduced, for example, by introducing a coding nucleic acid into the genetic material of the host, such as integration into a host chromosome, or as a non-chromosomal genetic material such as a plasmid. The term “exogenous”, when used in reference to expression of a coding nucleic acid, refers to introduction of the coding nucleic acid in an expressible form into a cell. The term “exogenous”, when used in reference to biosynthetic activity, refers to an activity that is introduced into a host parent cell. The source may be, for example, a homologous or heterologous coding nucleic acid that causes the referenced activity following introduction into the host parent cell. The term “endogenous” refers to a referenced molecule or activity that is present in the host cell prior to a given genetic modification. Similarly, the term “endogenous”, when used in reference to expression of a coding nucleic acid, refers to expression of a coding nucleic acid contained in a cell prior to a given genetic modification. The term “heterologous” refers to a molecule or activity derived from a source other than the referenced species, whereas “homologous” refers to a molecule or activity derived from or native to the host parent cell. Accordingly, exogenous expression of a coding nucleic acid may utilize either or both of heterologous and homologous coding nucleic acids.
As used herein, the term “genetic engineering” or “genetically engineered” refers to action of introducing one or more genetic modifications into a cell or a cell containing genetic modifications.
As used herein, the term “lactate” refers to “lactic acid” or a salt thereof.
An embodiment provides a recombinant yeast cell having decreased activity of RGT1 protein and an increased ability to produce a glycolytic intermediate or a glycolytic intermediate-derived substance, compared to a parent cell, in which the yeast cell includes a genetic modification that decreases RGT1 activity.
In the yeast cell, RGT1 protein may be a glucose-responsive transcription factor or a glucose transport transcription regulator which regulates the expression of several glucose transporter (HXT) genes in response to glucose. RGT1 may be also referred to as “restores glucose transport protein 1”. RGT1 associates with Mth1/Std1 to repress hexose transporter (HXT) expression at low levels of glucose, and high levels of glucose cause PKA to repress HXT expression. The hexose transporter refers to any protein such as enzyme, which can translocate a hexose such as glucose or fructose across the plasma membrane. The hexose transporter may be exemplified by HXT1, HXT2, HXT3, HXT4, HXT5, HXT6, HXT7, HXT8, HXT9, HXT10, HXT11, HXT12, HXT13, HXT14, HXT15, HXT16, HXT17, GAL2, SNF3, or RGT2. RGT1 protein may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to an amino acid sequence of SEQ ID NO. 1, or a yeast homologue thereof. RGT1 protein may be encoded by a polynucleotide sequence of SEQ ID NO. 2. RGT1 gene may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a polynucleotide sequence encoding the amino acid of SEQ ID NO. 1. The RGT1 gene may have a polynucleotide sequence (NM_001179604.3) of SEQ ID NO. 2.
The yeast cell having a modification of deleting or disrupting the gene encoding RGT eliminates repression of HXT expression via association with Mth1/Std1, and increases glucose transport by constitutive expression of HXT at low levels of glucose. Further, the yeast cell induces HTX expression to increase glucose transport irrespective of PKA activity at high levels of glucose.
In the yeast cell, an endogenous gene encoding RGT1 protein may be disrupted. In the yeast cell, the endogenous gene encoding RGT1 protein may be disrupted so that its expression is low enough to decrease the RGT1 protein activity, compared to its parent cell.
In the yeast cell, the yeast cell may further include a genetic modification that decreases HXK2 activity, in which the HXK2 protein activity is decreased, compared to the parent cell.
HXK2 protein may be hexokinase isoenzyme 2. HXK2 protein may be an enzyme belonging to EC. 2.7.1.1. HXK2 protein may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to an amino acid sequence of SEQ ID NO. 3, or a yeast homologue thereof. HXK2 protein may be encoded by a polynucleotide sequence of SEQ ID NO. 4. HXK2 gene may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a polynucleotide sequence encoding the amino acid of SEQ ID NO. 3. HXK2 may have a polynucleotide sequence of SEQ ID NO. 4.
In the yeast cell, the endogenous gene encoding HXK2 protein may be disrupted. In the yeast cell, the endogenous gene encoding HXK2 protein may be disrupted so that its expression is low enough to decrease the HXK2 protein activity, compared to its parent cell.
The yeast cell may have an ability to consume glucose at an increased glucose consumption rate, compared to the parent cell. The glucose consumption may be a process of forming two molecules of pyruvic acid from one molecule of glucose via glycolysis. The yeast cell may have an increased ability to produce a glycolytic intermediate or a glycolytic intermediate-derived substance, compared to a non-genetically engineered cell or a parent cell. The production may be production of a glycolytic intermediate or a glycolytic intermediate-derived substance within cells or secretion of a glycolytic intermediate or a glycolytic intermediate-derived substance after production within cells.
As used herein, the term “derived substance” may be a substance formed from a particular substance by a biosynthetic process. The “glycolytic intermediate-derived substance” may be included in the glycolytic intermediate. And, for example, the glycolytic intermediate-derived substance may be a substance formed from a pyruvate, by a biosynthetic process. The “biosynthetic process” includes a biosynthetic process that naturally exists in cells as well as a biosynthetic process that is newly created by introduction of a foreign gene. Specifically, the glycolytic intermediate may be glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), fructose-1,6-bisphosphate (FBP), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate (PEP), or pyruvate. The glycolytic intermediate-derived substance may be a DHAP-derived substance, a glyceraldehyde-3-phosphate (GAP)-derived substance, or a pyruvate-derived substance. The “DHAP-derived substance” may be glycerol-3-phosphate (G3P), glycerol, a glycerol-derived product, or a combination thereof. The “pyruvate-derived substance” may be alcohol, organic acid, or a combination thereof. The “pyruvate-derived substance” may be ethanol, acetic acid, acetyl-CoA, lactate, a TCA cycle intermediate, a product derived therefrom, or a combination thereof. The TCA cycle intermediate may be citric acid, itaconic acid, isocitrate, oxalosuccinate, alpha-ketoglutarate, succinic acid, succinyl-CoA, fumaric acid, malate, oxaloacetate, or a combination thereof. The substance derived from the TCA cycle intermediate may be a substance derived from succinic acid. The substance derived from the TCA cycle intermediate may be succinyl-CoA, succinic semialdehyde (SSA), 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, 4-hydroxybutylaldehyde, 1,3-butanediol (1,3-BDO), 1,4-butanediol (1,4-BDO), butanol, or isobutanol. The yeast cell may include a gene encoding an enzyme that functions to convert succinic acid to 1,4-BDO. The enzyme may be exemplified by CoA-dependent succinate semialdehyde dehydrogenase, 4-hydroxybutyrate (4-HB) dehydrogenase, 4-hydroxybutyryl-CoA (4HB-CoA) transferase, aldehyde/alcohol dehydrogenase, and Clostridium acetobutylicum AdhE2.
The yeast cell may be a strain belonging to Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Shizosaccharomyces, Issachenkia, or Hansenula, for example, Saccharomyces cerevisiae (S. cerevisiae), Saccharomyces bayanus (S. bayanus), Saccharomyces boulardii (S. boulardii), Saccharomyces bulderi (S. bulderi), Saccharomyces cariocanus (S. cariocanus), Saccharomyces cariocus (S. cariocus), Saccharomyces chevalieri (S. chevalieri), Saccharomyces dairenensis (S. dairenensis), Saccharomyces ellipsoideus (S. ellipsoideus), Saccharomyces eubayanus (S. eubayanus), Saccharomyces exiguus (S. exiguus), Saccharomyces florentinus (S. florentinus), Saccharomyces kluyveri (S. kluyveri), Saccharomyces martiniae (S. martiniae), Saccharomyces monacensis (S. monacensis), Saccharomyces norbensis (S. norbensis), Saccharomyces paradoxus (S. paradoxus), Saccharomyces pastorianus (S. pastorianus), Saccharomyces spencerorum (S. spencerorum), Saccharomyces turicensis (S. turicensis), Saccharomyces unisporus (S. unisporus), Saccharomyces uvarum (S. uvarum), or Saccharomyces zonatus (S. zonatus).
The yeast cell may have an increased activity of an enzyme that exists in a synthetic pathway of a pyruvate-derived substance from pyruvate, a synthetic pathway of glycerol from DHAP, or a synthetic pathway of a glycerol-derived substance from glycerol. The synthetic pathway of glycerol from DHAP may include G3P dehydrogenase (GPDH) that catalyzes conversion of DHAP and NADH to G3P and NAD+, and G3Pase that catalyzes conversion of G3P to glycerol and Pi.
The “pyruvate-derived substance” is the same as described above. The increase may be caused by an increase in expression of a polynucleotide encoding the enzyme. The yeast cell may have an increased activity of an enzyme that converts pyruvate to lactate, or an increased activity of an enzyme that converts pyruvate to ethanol. The increase may be caused by an increase in expression of a polynucleotide that encodes the enzyme converting pyruvate to lactate or the enzyme converting pyruvate to ethanol. The polynucleotide that encodes the enzyme converting pyruvate to lactate may be a polynucleotide encoding an enzyme classified as EC 1.1.1.27 or EC 1.1.1.28. The enzyme in the pathway of converting pyruvate to ethanol may be one or more of pyruvate decarboxylase and alcohol dehydrogenase. The pyruvate decarboxylase may be an enzyme classified as EC 4.1.1.1. The alcohol dehydrogenase (ADH) may be an enzyme classified as EC. 1.1.1.2.
In the above aspect, the enzyme that catalyzes the conversion of pyruvate to lactate may be lactate dehydrogenase (LDH) belonging to EC 1.1.2.27 or EC 1.1.1.28. The LDH may be NAD(P)H-dependent. Further, LDH may act on D-lactate and/or L-lactate. The LDH may have a sequence identity of 95% or more to an amino acid sequence of SEQ ID NO. 5. SEQ ID NO. 5 represents LDH of Pelodiscus sinensis japonicus. 
The yeast cell may include an exogenous gene encoding an enzyme that catalyzes conversion of pyruvate to lactate. The exogenous LDH gene may be expressed enough to increase the activity of the enzyme that catalyzes conversion of pyruvate to lactate, compared to its parent cell. The exogenous LDH gene may code for an amino acid sequence having a sequence identity of 95% or more to the amino acid sequence of SEQ ID NO. 5. The exogenous LDH gene may have a sequence identity of 95% or more to a nucleotide sequence of SEQ ID NO. 6. The LDH exogenous gene may have a sequence, which is changed to have codons suitable for expression in the yeast cell, or a sequence having optimized codons. This codon alteration may be properly performed within the range of causing no change in the amino acid sequence of the protein. SEQ ID NO. 6 represents LDH gene of Pelodiscus sinensis japonicus. 
The exogenous LDH gene may be included in the genome of a cell. The exogenous LDH gene may code for an enzyme that acts on L-Ldh, D-Ldh or both of them. Therefore, the yeast cell may produce L-lactic acid or D-lactic acid or a racemic mixture thereof, or a salt thereof. The exogenous LDH gene may include those derived from bacteria, yeasts, fungi, and animals, for example, rodents, mammals, amphibians and Sauropsida. The exogenous LDH gene may be a polynucleotide encoding one or more of LDH selected from the Pelodiscus sinensis japonicus, Ornithorhynchus anatinus, Tursiops truncatus, Rattus norvegicus, or Xenopus laevis. The lactate dehydrogenase derived from Pelodiscus sinensis japonicus, Ornithorhynchus anatinus, Tursiops truncatus, Rattus norvegicus, or Xenopus laevis may have an amino acid sequence of SEQ ID NO. 5, 7, 8, or 9, respectively. The lactate dehydrogenase may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to the amino acid sequence of SEQ ID NO. 5, 7, 8, or 9, respectively. The gene encoding the lactate dehydrogenase may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a nucleotide sequence of SEQ ID NO. 6, 10, 11, or 12.
The exogenous LDH gene may be expressed from a vector including the same. The vector may include a replication origin, a promoter, a polynucleotide encoding LDH, 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 CCW12 promoter, a CYC promoter, a TEF1 promoter, a PGK1 promoter, a GPD promoter, and an ADH promoter. The CCW12 promoter, CYC promoter, TEF1 promoter, PGK1 promoter, GPD promoter, or ADH promoter may have A polynucleotide sequence of SEQ ID NO. 13, 14, 15, 16, 17, or 18, respectively. The terminator may be selected from the group consisting of PGK1, CYC1, and GAL1. The CYC1 terminator may have a polynucleotide sequence of SEQ ID NO. 19. The vector may further include a selection marker.
The yeast cell may include one copy or multiple copies of an LDH gene, for example, about 2 to about 10 copies. The yeast cell may include, for example, about 1 to about 10, 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, 1 about to about 3, about 2 to 1 about 0, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2 to about 4, or about 2 to about 3 copies of LDH gene(s). If the yeast cell includes multiple LDH genes, each of the genes may include copies of the same gene or copies of two or more different LDH genes. The multiple copies of exogenous LDH genes may be included in the same locus or multiple loci in the genome of a host cell.
An example of the yeast cell may be a yeast cell having an ability to produce lactate, in which RGT1 gene is disrupted and the exogenous gene encoding the enzyme (LDH) that catalyzes the conversion of pyruvate to lactate is included, compared to the parent cell.
Another example of the yeast cell may be a yeast cell having an ability to produce lactate, in which RGT1 gene and HXK2 gene are disrupted and the exogenous gene encoding the enzyme (LDH) that catalyzes the conversion of pyruvate to lactate is included, compared to the parent cell.
In the above aspect, the yeast cell may further include a genetic modification of decreasing an activity of an enzyme that catalyzes conversion of acetaldehyde to ethanol, and therefore, the yeast cell has a decreased activity of the enzyme that catalyzes conversion of acetaldehyde to ethanol.
The enzyme that catalyzes the conversion of acetaldehyde to ethanol may be ADH belonging to EC 1.1.1.1. Examples of ADH may include ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7. The ADH may be NADH-dependent. The ADH1 gene and ADH1 protein may have a polynucleotide sequence of SEQ ID NO. 20 and an amino acid sequence of SEQ ID NO. 21, respectively.
In the yeast cell, an endogenous gene encoding the enzyme that catalyzes conversion of acetaldehyde to ethanol may be disrupted. In the yeast cell, the gene may be disrupted so that its expression is low enough to decrease the activity of the enzyme that catalyzes conversion of acetaldehyde to ethanol, compared to its parent cell.
The yeast cell according to the above aspect may further include a genetic modification of decreasing an activity of an enzyme that catalyzes the conversion of pyruvate to acetaldehyde, an enzyme that catalyzes the conversion of lactate to pyruvate, an enzyme that catalyzes the conversion of DHAP to G3P, an enzyme that catalyzes the conversion of G3P to glycerol, an enzyme that catalyzes the conversion of acetaldehyde to acetate, or a combination thereof.
In the yeast cell, the enzyme that catalyzes the conversion of pyruvate to acetaldehyde may belong to EC 4.1.1.1, the enzyme that catalyzes the conversion of lactate to pyruvate may belong to EC 1.1.2.4 or EC 1.1.2.3, the enzyme that catalyzes the conversion of DHAP to G3P may belong to EC 1.1.1.8, the enzyme that catalyzes the conversion of G3P to glycerol may belong to EC 3.1.3.21, and the enzyme that catalyzes the conversion of acetaldehyde to acetate may belong to EC 1.2.1.3, EC 1.2.1.4, or EC 1.2.1.5.
In the yeast cell, a gene encoding the enzyme that catalyzes the conversion of pyruvate to acetaldehyde, a gene encoding the enzyme that catalyzes the conversion of lactate to pyruvate, a gene encoding the enzyme that catalyzes the conversion of DHAP to G3P, a gene encoding the enzyme that catalyzes the conversion of G3P to glycerol, a gene encoding the enzyme that catalyzes the conversion of acetaldehyde to acetate, or a combination thereof may be disrupted.
The enzyme that catalyzes the conversion of pyruvate to acetaldehyde may be pyruvate decarboxylase (PDC). PDC may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to an amino acid sequence of SEQ ID NO. 22. The PDC gene may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a polynucleotide sequence of SEQ ID NO. 23. The PDC includes PDC1 (SEQ ID NO. 23), PDC5, and PDC6. The PDC may catalyze conversion of pyruvate to acetaldehyde under anaerobic or aerobic conditions. The PDC gene may be disrupted by replacement with LDH gene. In the yeast cell, the PDC gene may be attenuated. In the yeast cell, one or more of PDC1 gene, PDC5 gene, and PDC6 gene may be also attenuated. In the yeast cell, the gene(s) encoding one or two of PDC1, PDC5, and PDC6 may be inactivated. For example, PDC1 gene, PDC5 gene, PDC6 gene, PDC1 gene and PDC5 gene, PDC1 gene and PDC6 gene, or PDC5 gene and PDC6 gene may be inactivated.
The enzyme that catalyzes the conversion of lactate to pyruvate may be lactate cytochrome-c oxidoreductase (CYB2). The enzyme that catalyzes the conversion of lactate to pyruvate may be cytochrome c-dependent. The CYB2 may belong to EC 1.1.2.4 which acts on D-lactate, or EC 1.1.2.3 which acts on L-lactate. The enzyme that catalyzes the conversion of lactate to pyruvate may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to an amino acid sequence of SEQ ID NO. 24. A gene that codes for the enzyme that catalyzes the conversion of lactate to pyruvate may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a polynucleotide sequence of SEQ ID NO. 25. The CYB2 gene may be disrupted by replacement with LDH gene.
The enzyme that catalyzes the conversion of DHAP to G3P may be NAD-dependent dependent glycerol-3-phosphate dehydrogenase (GPD). The GPD may be a NAD+-dependent enzyme. An example of the GPD may include cytosolic glycerol-3-phosphate dehydrogenase, which is an enzyme catalyzing reduction of dehydroxyacetone phosphate (DHAP) to glycerol-3-phosphate using oxidation of NADH to NAD+. The GPD may belong to EC 1.1.1.8. Examples of the GPD include GPD1 and GPD2. The GPD may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to an amino acid sequence of SEQ ID NO. 26. A gene encoding the GPD may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a polynucleotide sequence of SEQ ID NO. 27. The GPD gene may be disrupted by replacement with LDH gene.
The enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol may be glycerol phosphate phosphatase (GPP). The GPP may be GPP1 and GPP2 derived from S. cerevisiae. The GPP may have a nucleotide sequence of SEQ ID NO. 28 and an amino acid sequence of SEQ ID NO. 29. The GPP gene may be disrupted by replacement with LDH gene.
The enzyme that catalyzes the conversion of acetaldehyde to acetate may be acetaldehyde dehydrogenase (ALD). The ALD may be NAD(P)+-dependent. The ALD may have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to an amino acid sequence of SEQ ID NO. 30. The ALD gene have a sequence identity of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% to a polynucleotide sequence of SEQ ID NO. 31. The ALD includes ALD6 (also called ALD1), ALD2, ALD3, ALD4, and ALD5. The ALD gene may be disrupted by replacement with LDH gene. In the yeast cell, the ALD gene may be attenuated. In the yeast cell, one or more of ALD6 gene, ALD2 gene, and ALD3 gene may be also attenuated. In the yeast cell, one or two of ALD6 gene, ALD2 gene, and ALD3 gene may be inactivated.
In the yeast cell, the enzyme that catalyzes the conversion of pyruvate to acetaldehyde may be PDC, the enzyme that catalyzes the conversion of lactate to pyruvate may be CYB2, the enzyme that catalyzes the conversion of DHAP to G3P may be NAD-dependent GPD, the enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol may be GPP, and the enzyme that catalyzes the conversion of acetaldehyde to acetate may be acetaldehyde dehydrogenase.
The yeast cell according to an aspect may have an increased activity of an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA, compared to its parent cell.
The enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA may be acylating acetaldehyde dehydrogenase (A-ALD) belonging to EC 1.2.1.10.
A type of the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA may be a part of a bifunctional aldolase-dehydrogenase complex involved in 4-hydroxy-2-ketovalerate catabolism. Such bifunctional enzyme catalyzes the final two steps of the meta-cleavage pathway for catechol, which is an intermediate in many bacterial species in the degradation of phenol, toluene, naphthalene, biphenyl and other aromatic compounds. 4-Hydroxy-2-ketovalerate is first converted by 4-hydroxy-2-ketovalerate aldolase to pyruvate and acetaldehyde, subsequently acetaldehyde is converted by ADA to acetyl-CoA. An example of this type of A-ALD is DmpF in Pseudomonas sp CF600 (Genbank No: CAA43226). MphF protein of E. coli is homologous to the DmpF protein. Another type thereof is a protein that catalyzes the reversible conversion of acetyl-CoA to acetaldehyde in strictly or facultative anaerobic microorganisms, but does not possess ADH activity. An example of this type of protein may be a protein that is reported in Clostridium kluyveri. A-ALD has been annotated in the genome of Clostridium kluyveri DSM 555 (GenBank No: EDK33116). A homologous protein AcdH was identified in the genome of Lactobacillus plantarum (GenBank No: NP-784141). Another example of this type of protein is a product of the gene in Clostridium beijerinckii NRRL B593 (Genbank No: AAD31841). An example of A-ALD may be E. coli-derived MhpF or a functional homologue thereof, for example, E. coli and S. typhimurium-derived EutE (e.g., EutE gene having a polynucleotide sequence of SEQ ID NO. 32 and EutE protein having an amino acid sequence of SEQ ID NO. 33), or Pseudomonas sp. CF600-derived dmpF. The A-ALD may be NAD(P)+-dependent. The A-ALD may have an activity of catalyzing the following reaction:

Acetaldehyde+CoA (coenzyme A)+NAD+<=>acetyl-CoA+NADH+H+

The A-ALD may be expressed without formation of a complex with other proteins. The yeast cell may not include, for example, an exogenous enzyme belonging to EC 4.1.3.39 or a gene thereof.
The A-ALD may be derived from E. coli. In E. coli, the A-ALD gene, namely, mhpF, may be one of the units consisting of the transcription units, mhpA, mhpB, mhpC, mhpD, mhpE, and mhpF. Generally, MhpE and MhpF exist as a complex in other microorganisms, but MhpF may exist singly and shows its activity in E. coli. In this regard, the enzyme MhpF, which catalyzes conversion of acetaldehyde to acetyl-CoA, may have a sequence identity of 95% or more to an amino acid sequence of SEQ ID NO. 34.
The yeast cell may include an exogenous gene encoding the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA. In the yeast cell, the exogenous A-ALD gene may be expressed enough to increase the activity of the enzyme that catalyzes conversion of acetaldehyde to acetyl-CoA, compared to its parent cell. The exogenous A-ALD gene may code for an amino acid having a sequence identity of 95% or more to an amino acid sequence of SEQ ID NO. 34. The exogenous A-ALD gene may have a sequence identity of 95% or more to a nucleotide sequence of SEQ ID NO. 35 or SEQ ID NO. 36. SEQ ID NO. 35 represents a nucleotide sequence of the A-ALD gene derived from E. coli. The exogenous A-ALD gene may have a sequence, which is changed to have codons suitable for expression in the yeast cell, or a sequence having optimized codons. This codon alteration may be properly performed within the range of causing no change in the amino acid sequence of the protein. SEQ ID NO. 36 represents an example of the nucleotide sequence having yeast-optimized codons of the E. coli-derived A-ALD gene.
The exogenous gene may be introduced into the parent cell using an expression vector. Further, the exogenous gene may be introduced in the form of a linear polynucleotide into the parent cell. Furthermore, the exogenous gene may be expressed from the expression vector (e.g., plasmid) within the cell. For stable expression, the exogenous gene may be expressed by integration into a genetic material (e.g., chromosome) within the cell. The exogenous gene may be appropriately regulated by an exogenous promoter operably linked to the gene. The promoter may be a promoter derived from ccw12, pdc1, tef1 or pgk1 gene.
Another aspect provides a method of producing the yeast cell having an ability to produce lactate, the method including: introducing a gene encoding the enzyme that catalyzes conversion of pyruvate to lactate; and disrupting the gene encoding RGT1 protein in the yeast cell.
The method of producing the yeast cell having an ability to produce lactate may further include disrupting the gene encoding HXK2 protein in the yeast cell.
In the method of producing the yeast capable of producing lactate, introducing the yeast cell with the gene encoding the enzyme that catalyzes conversion of pyruvate to lactate is the same as follows: In this step, the “yeast cell”, the “enzyme that catalyzes the conversion of pyruvate to lactate” and the “gene encoding the enzyme that catalyzes the conversion of pyruvate to lactate” are the same as described above.
The “gene encoding the enzyme that catalyzes the conversion of pyruvate to lactate” may be introduced to be integrated into an endogenous genetic material (e.g., chromosome) of the yeast cell. In this case, the genes are inserted into one or more positions of the particular gene of the endogenous genetic material (e.g., chromosome) of the yeast cell, resulting in disruption of the one or more genes. The particular gene may include the gene encoding the enzyme that catalyzes the conversion of pyruvate to acetaldehyde, the gene encoding the enzyme that catalyzes the conversion of lactate to pyruvate, the gene encoding the enzyme that catalyzes the conversion of DHAP to G3P, the gene encoding the enzyme that catalyzes the conversion of G3P to glycerol, and the gene encoding the enzyme that catalyzes the conversion of acetaldehyde to ethanol. The particular gene may include PDC, CYB2, GPD, GPP, and ADH genes.
The gene may exist within the yeast cell without integration into the endogenous genetic material of the yeast cell. In this case, the gene may be included in a vector such as a plasmid and may exist separately from the endogenous genetic material of the yeast cell.
The gene may be introduced in an expressible form into the yeast cell, and then expressed to produce its gene product, the “enzyme that catalyzes the conversion of pyruvate to lactate” in the yeast cell. The expressible form may be in the form of operably linking the gene to its expression regulatory sequence. For example, the gene may be operably linked to one or more of an exogenous enhancer, operator, promoter, and transcription terminator, and thus, it may become expressible as it is in the yeast cell, or it may be linked to a regulatory sequence of the yeast cell to be expressible. The promoter may be selected from the group consisting of a CCW12 promoter, a CYC promoter, a TEF1 promoter, a PGK1 promoter, a GPD promoter, and an ADH promoter. The CCW12 promoter, CYC promoter, TEF1 promoter, PGK1 promoter, GPD promoter, and ADH promoter may have the polynucleotide sequence of SEQ ID NOs. 13, 14, 15, 16, 17, and 18, respectively. 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. 19. The vector may further include a selection marker.
The introducing may be performed by introducing a genetic material into a yeast cell known in the art (R. Danile Gietz et al., Biotechniques 30:816-831, April 2001). The introducing method may include a spheroplast method, intact yeast cell transformation, and electroporation. In the intact yeast cell transformation, specific monovalent alkali cations (Na+, K+, Rb+, Cs+ and Li+) may be used in combination with PEG to stimulate plasmid DNA uptake by intact yeast cells. For example, heat shock may be applied to an aqueous solution containing PEG, LiAc, carrier ssDNA, and plasmid DNA. Electroporation may involve applying an electric pulse to a mixed medium containing the yeast cell and DNA such as plasmid DNA.
Therefore, the introducing step may include contacting the yeast cell with the gene encoding the enzyme that catalyzes the conversion of pyruvate to lactate in an appropriate liquid medium. The yeast cell may be a spheroplast, or an intact yeast cell. The liquid medium may differ depending on the selected transformation method. The liquid medium may be, for example, water, an aqueous solution, or a buffer. The aqueous medium may include monovalent alkali cations (one or more of Na+, K+, Rb+, Cs+ and Li+) and PEG. The aqueous medium may include carrier ssDNA. The aqueous medium may be an aqueous solution containing PEG, LiAc, and carrier ssDNA.
The contacting may be performed by applying heat shock or electric pulse to the yeast cell and the gene. The heat shock may be incubation at about 40 to about 45° C., for example, about 42° C. The electric pulse may be applied either in an electroporation cuvette or between electrodes in a petri dish. The parameters of the electroporation, field strength (kV/cm), capacitance (uF), and resistance may differ depending on details of cell preparation. Therefore, transformation efficiency may vary depending on the yeast strain. For any given yeast strain, those skilled in the art investigate the parameters of the pulse according to the cell and select proper parameters to obtain a sufficient number of transformants.
In the introducing, the both genes are included in a vector, together with a homologous sequence to an endogenous genetic material of the parent yeast cell. The homologous sequence is complementary to a target sequence which is present in the endogenous genetic material of the parent yeast cell, and thus it may be substituted for the target sequence by homologous recombination. The target sequence includes the gene encoding the enzyme that catalyzes conversion of pyruvate to acetaldehyde, the gene encoding the enzyme that catalyzes conversion of lactate to pyruvate, the gene encoding the enzyme that catalyzes conversion of DHAP to G3P, the gene encoding the enzyme that catalyzes conversion of G3P to glycerol, and the gene encoding the enzyme that catalyzes conversion of acetaldehyde to ethanol. The particular gene includes PDC, CYB2, GPD, GPP, and ADH genes. The vector may include two sequences that are homologous to the 5′-terminal region and the 3′-terminal region of the target sequence, respectively. In this case, the introducing may include incubation of the yeast cell under a selection pressure during or after the contacting. The selection pressure refers to a substance or state that is forced to select only the cells in which a homologous recombination occurs. The selection pressure includes incubation in the presence of an antibiotic. In this case, the vector may include a gene encoding an enzyme that degrades the antibiotic.
In the method of producing the yeast capable of producing lactate, disrupting the respective gene(s) encoding RGT1 protein or RGT1 protein and HXK2 protein in the yeast cells is the same as follows:
The disrupting may include contacting the yeast cell with a polynucleotide including a homologous sequence to the gene encoding RGT1 protein, or the respective genes encoding RGT1 protein and HXK2 protein in an appropriate liquid medium. The homologous sequence may be homologous to the entire or a part of the gene. The homologous sequence may be homologous to a coding region or an expression regulatory region of the gene. The polynucleotide including the homologous sequence to the gene may be linked to other genes, for example, a gene encoding the enzyme that is involved in biosynthesis of lactate, such as an LDH gene. The polynucleotide including the homologous sequence to the gene may be included in a vector such as a plasmid. The homologous sequence may be used for replacement of the respective gene(s) encoding RGT1 protein or RGT1 protein and HXK2 protein by homologous recombination. The vector may include two sequences that are homologous to the 5′-terminal region and the 3′-terminal region of the target sequence, respectively. In this case, the disrupting may include incubation of the yeast cell under a selection pressure during or after the contacting. The selection pressure refers to a substance or state that provides for the selection of only those cells in which a homologous recombination occurs. The selection pressure includes incubation in the presence of an antibiotic. In this case, the vector may include a gene encoding an enzyme that degrades the antibiotic.
In the disrupting, the contacting may be performed under the same conditions as a method of introducing a genetic material into a yeast cell, which is known in the art, unless otherwise specified. The introduction method may include a spheroplast method, intact yeast cell transformation, and electroporation. In the intact yeast cell transformation, specific monovalent alkali cations (Na+, K+, Rb+, Cs+, and Li+) may be used in combination with PEG to stimulate plasmid DNA uptake by intact yeast cells. For example, heat shock may be applied to an aqueous solution containing PEG, LiAc, carrier ssDNA, and plasmid DNA. Electroporation may involve applying an electric pulse to a mixed medium containing the yeast cell and DNA such as plasmid DNA.
The yeast cell may be a spheroplast or an intact yeast cell. The liquid medium may differ depending on the selected transformation method. The liquid medium may be, for example, water, an aqueous solution, or a buffer. The aqueous medium may include monovalent alkali cations (one or more of Na+, K+, Rb+, Cs+, and Li+) and PEG. The aqueous medium may include carrier ssDNA. The aqueous medium may be an aqueous solution containing PEG, LiAc, and carrier ssDNA.
The contacting may be performed by applying heat shock or electric pulse to the yeast cell and the gene. The heat shock may be incubation at about 40 to about 45° C., for example, about 42° C. The electric pulse may be applied either in an electroporation cuvette or between electrodes in a petri dish. The parameters of the electroporation, field strength (kV/cm), capacitance (uF), and resistance may differ depending on details of cell preparation. Therefore, transformation efficiency may vary depending on the yeast strain. For any given yeast strain, those skilled in the art investigate the parameters of the pulse according to the cell and select proper parameters to obtain a sufficient number of transformants.
In the contacting, the respective genes are included in a vector, together with a homologous sequence to an endogenous genetic material of the parent yeast cell. The homologous sequence is complementary to a target sequence which is present in the endogenous genetic material of the parent yeast cell, and thus, it may be substituted for the target sequence by homologous recombination. The target sequence includes the respective gene(s) encoding RGT1 protein or RGT1 protein and HXK2 protein. Further, the target sequence includes the gene encoding the enzyme that catalyzes conversion of pyruvate to acetaldehyde, the gene encoding the enzyme that catalyzes conversion of lactate to pyruvate, the gene encoding the enzyme that catalyzes conversion of DHAP to G3P, the gene encoding the enzyme that catalyzes conversion of glycerol-3-phosphate to glycerol, and the gene encoding the enzyme that catalyzes conversion of acetaldehyde to ethanol. The particular gene includes PDC, CYB2, GPD, GPP, and ADH genes. The vector may include two sequences that are homologous to the 5′-terminal region and the 3′-terminal region of the target sequence, respectively. In this case, incubation of the yeast cell under a selection pressure during or after the contacting may be included. The selection pressure refers to a substance or state that is forced to select only the cells in which a homologous recombination occurs. The selection pressure includes incubation in the presence of an antibiotic. In this case, the vector may include a gene encoding an enzyme that degrades the antibiotic.
Still another aspect provides a method of producing the glycolytic intermediate or the glycolytic intermediate-derived substance, the method including: culturing the yeast cell in a cell culture medium to obtain a culture, whereby the yeast cell produces the glycolytic intermediate or the glycolytic intermediate-derived substance; and recovering the glycolytic intermediate or the glycolytic intermediate-derived substance from the culture.
This method includes culturing the yeast cell. The “yeast cell” is the same as described above.
The culturing may be performed in a medium containing a carbon source, for example, glucose. The medium used for culturing the yeast cell may be any general medium that is suitable for host cell growth, such as a minimal or complex medium containing proper supplements. The suitable medium may be commercially available or prepared by a known preparation method. The medium used for the culturing may be a medium that satisfies the requirements of a particular yeast cell. The medium may be a medium selected from the group consisting of a carbon source, a nitrogen source, a salt, trace elements and combinations thereof.
The culturing conditions may be properly controlled in order to obtain the glycolytic intermediate or the glycolytic intermediate-derived substance, for example, a pyruvate-derived substance such as pyruvate or lactate from the genetically engineered yeast cell. For proliferation, the cell may be cultured under aerobic conditions. Thereafter, the cell may be cultured under microaerobic conditions or anaerobic conditions in order to produce the glycolytic intermediate or the glycolytic intermediate-derived substance. The term “anaerobic conditions” means oxygen deficient conditions. The term “microaerobic conditions”, when used in reference to culture or growth conditions, means that a concentration of dissolved oxygen (DO) in a medium is more than 0% and less than about 10% of saturation for DO in a liquid medium. The microaerobic conditions also include growing or resting cells in a liquid medium or on a solid agar plate inside a sealed chamber which is maintained with an atmosphere of less than 1% oxygen. The percentage of oxygen may be maintained by, for example, sparging the culture with an N2/CO2 mixture or other suitable non-oxygen gas. The oxygen conditions include maintaining the concentration of DO at about 0% to about 10%, for example, about 0 to about 8%, about 0 to about 6%, about 0 to 4%, or about 0 to about 2%.
The term “culture conditions” means conditions for culturing the yeast cell. Such culture conditions may include, for example, a carbon source, a nitrogen source, or an oxygen condition utilized by the yeast cell. The carbon source may be an assimilable carbon source that can be assimilated by any yeast cell. The carbon source may include monosaccharides, disaccharides, or polysaccharides. The carbon source may be glucose, fructose, mannose, or galactose. The nitrogen source that can be utilized by the yeast cell may be an organic nitrogen compound or an inorganic nitrogen compound. The nitrogen source may be exemplified by amino acids, amides, amines, nitrates, or ammonium salts.
The medium may include a buffering agent that maintains a pH at about 3 to about 5. The buffering agent may be bicarbonate. The culturing may be performed by maintaining a pH of the medium at about 3 to about 5. The pH of the medium may be maintained at about 3 to about 5 by including the buffering agent in the medium or by adding an acid or a base to the medium during the culturing. For example, by monitoring the pH of the medium, a acid is added to the medium at a pH that is higher than about 5, and an base is added to the medium at a pH that is lower than about 3.
The culture may include the glycolytic intermediate or the glycolytic intermediate-derived substance, for example, the pyruvate-derived substance such as pyruvate or lactate.
Isolation of the glycolytic intermediate or the glycolytic intermediate-derived substance, for example, the pyruvate-derived substance such as pyruvate or lactate, from the culture may be performed by a general method known in the art. Such isolation method may be centrifugation, filtration, ion chromatography, or crystallization. For example, the culture is centrifuged at a low speed to remove biomass, and a resulting supernatant is subjected to ion chromatography for isolation.
The recombinant yeast cell according to an aspect is able to effectively produce the glycolytic intermediate or the glycolytic intermediate-derived substance.
The method of producing the recombinant yeast cell according to another aspect is used to effectively produce the glycolytic intermediate or the glycolytic intermediate-derived substance.
The method of effectively producing the glycolytic intermediate or the glycolytic intermediate-derived substance according to still another aspect is used to effectively produce the glycolytic intermediate or the glycolytic intermediate-derived substance by the recombinant yeast cell.
Hereinafter, the present invention will be described in more detail with reference to the exemplary embodiments. However, the exemplary embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation.
Example 1: Production of Lactate from RGT1 Gene-Disrupted Yeast Strain
1. Preparation of Strain
S. cerevisiae strain used in this Example is prepared as follows:
(1) Preparation of S. cerevisiae CEN.PK2-1D (Δadh1::ldh)
(1.1) Preparation of adh1 Gene Deletion Cassette
In order to prepare an ‘ldh cassette’-containing vector, PCR is performed using genomic DNA of S. cerevisiae CEN.PK2-1D as a template and a primer set of SEQ ID NOs. 37 and 38 as primers to amplify a CCW12 gene promoter (hereinafter, referred to as “P CCW12” or “CCW12 promoter”), and amplification products of the CCW12 gene promoter (SEQ ID NO. 13) and a synthesized ldh gene (SEQ ID NO. 6) (DNA2.0 Inc., USA) are cleaved with SacI/XbaI and BamHI/SalI, respectively and ligated with a pRS416 vector (ATCC87521TM), which is cleaved with the same enzymes, respectively. The pRS416 vector is a yeast centromere shuttle plasmid having a T7 promoter, an ampicillin resistance in bacteria, a URA3 cassette in yeast (selection marker), and restriction enzyme cloning sites.
Further, PCR is performed using a pCEP4 plasmid (invitrogen, Cat. no. V044-50) as a template and a primer set of SEQ ID NOs. 39 and 40 as primers to amplify an “HPH cassette” sequence (SEQ ID NO. 41). The amplified “HPH cassette” and the pRS416 vector are cleaved with SacI enzyme, and ligated with each other so as to prepare a p416-ldh-HPH vector containing a structure of operably linking the ‘ldh cassette’ and the “HPH cassette”. FIG. 2 shows a cleavage map of the p416-ldh-HPH vector. In FIG. 2, “P CCW12” and “C2 LDH” indicate the CCW12 promoter and LDH orf, respectively. The pCEP4 plasmid is an episomal mammalian expression vector that uses the cytomegalovirus (CMV) immediate early enhancer/promoter for high level transcription of recombinant genes inserted into the multiple cloning site. pCEP4 has a hygromycin B resistance gene for stable selection in transfected cells. Here, the ‘ldh cassette’ represents a region that allows the ldh gene to be expressed, because it contains the ldh gene and its regulatory region. Transcription of the ldh gene is allowed in the presence of the CCW12 promoter. Further, the ‘HPH (hygromycin B phosphotransferase) cassette’ represents a region that allows the hygromycin B resistance gene to be expressed, because it contains the hygromycin B resistance gene and its regulatory region.
An adh1 deletion cassette is prepared by PCR using the p416-ldh-HPH vector as a template and a primer set of SEQ ID NO. 42 and SEQ ID NO. 43 as primers. In SEQ ID NO. 42 and SEQ ID NO. 43, the sequence at position 1-51 represents a region which is substituted for the adh1 gene by homologous recombination with a homologous sequence in a genome of S. cerevisiae. 
The adh1 deletion cassette is further used for adh1 inactivation and ldh gene introduction by replacement of the ADH (adh1)-encoding gene with the ldh-HPH cassette in the genome of S. cerevisiae CEN.PK2-1D.
(1.2) Preparation of S. cerevisiae CEN.PK2-1D (Δadh1::ldh)
In order to replace the adh1 gene with the ldh gene in S. cerevisiae CEN.PK2-1D, the “adh1 deletion cassette” prepared in (1.1.2) is introduced into S. cerevisiae CEN.PK2-1D strain by heat shock transformation, and cultured in an YPD medium (Yeast extract 1 (w/v) %, peptone 1 (w/v) %, and glucose 2 (w/v) %) containing 200 ug/mL of hygromycin at 30° C. for 3 days for replacement of the chromosomal adh1 gene with the ldh gene, thereby preparing a S. cerevisiae CEN.PK2-1D (Δadh1::ldh) strain).
(2) Preparation of S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) Strain
(2.1) Construction of Vector for pdc1, cyb2, and gpd1 Deletions
To prepare a pyruvate decarboxylase 1 (Pdc1) deletion cassette, PCR is performed using p416-ldh-HPH as a template and a primer set of SEQ ID NOs. 44 and 45 as primers, and an amplification product is cleaved with SacI and ligated with a pUC57-Ura3HA vector (DNA2.0 Inc.: SEQ ID NO. 46) which is cleaved with the same enzyme, so as to prepare pUC57-ura3HA-ldh. Next, PCR is performed using pUC57-ura3HA-ldh as a template and a primer set of SEQ ID NOs. 47 and 48 as primers so as to prepare a pdc1 deletion cassette. In SEQ ID NO. 47 and SEQ ID NO. 48, each of the sequences at positions 1-42 and 1-44 represents a region which is substituted for the pdc1 gene by homologous recombination with a homologous sequence of S. cerevisiae. 
Further, L-lactate cytochrome-c oxidoreductase (cyb2) gene deletion cassette is amplified by PCR using the deletion vector pUC57-ura3HA-ldh as a template and a primer set of SEQ ID NOs. 49 and 50 as primers. In SEQ ID NO. 49 and SEQ ID NO. 50, the sequence at position 1-45 represents a region which is substituted for the cyb2 gene by homologous recombination with a homologous sequence of S. cerevisiae. 
Further, a glycerol-3-phosphate dehydrogenase (gpd1) gene deletion cassette is amplified by PCR using the deletion vector pUC57-ura3HA-ldh as a template and a primer set of SEQ ID NOs. 51 and 52 as primers. In SEQ ID NO. 52, the sequence at position 1-50 represents a region which is substituted for the gpd1 gene by homologous recombination with a homologous sequence of a chromosome of S. cerevisiae. 
(2.2) Preparation of S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) Strain
First, in order to replace the pdc1 gene with the ldh gene in S. cerevisiae CEN.PK2-1D (Δadh1::ldh), the “pdc1 deletion cassette” prepared in (2.1) is introduced into S. cerevisiae CEN.PK2-1D (Δadh1::ldh) prepared in (1) by heat shock transformation. After heat shock, the cells are cultured in a Ura-drop out medium as a selection marker at 30° C. for 3 days to replace the chromosomal pdc1 gene with the ldh gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 53 and 54 to examine deletion of the pdc1 gene.
As a result, S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh) strain is identified.
Next, to replace the cyb2 gene with the ldh gene in S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh) strain, the “cyb2 deletion cassette” prepared in (2.1) is introduced into the strain by heat shock transformation. After heat shock, the cells are cultured in a Ura-drop out medium as a selection marker at 30° C. for 3 days to replace the chromosomal cyb2 gene with the ldh gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 55 and 56 to examine deletion of the cyb2 gene.
As a result, S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh) strain is identified.
Next, to replace the gpd1 gene with the ldh in S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh) strain, the “gpd1 deletion cassette” prepared in (2.1) is introduced into the strain by heat shock transformation. After heat shock, the cells are cultured in a Ura-drop out medium as a selection marker at 30° C. for 3 days to replace the chromosomal gpd1 gene with the ldh gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 57 and 58 to examine deletion of the gpd1 gene.
As a result, S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) strain is identified.
(3) Preparation of S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, mhpF, Δald6, EutE) Strain
(3.1) Construction and Introduction of Vector for mhpF Insertion
To enhance a pathway of converting acetaldehyde to acetyl-CoA in S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh), an MhpF gene is inserted.
In detail, to express E. coli-derived MhpF gene (SEQ ID NO. 35) from a vector in S. cerevisiae, the MhpF gene (SEQ ID NO. 36) (DNA2.0 Inc., USA), which is codon-optimized for expression in S. cerevisiae, is synthesized. The synthesized MhpF gene has a TEF1 promoter sequence (SEQ ID NO. 15) at the 5′ end, and its transcription is regulated by the TEF1 promoter. pJ1214-mhpF (DNA2.0 Inc., USA) containing this synthesized TEF1 promoter-MhpF gene is provided. pJ1214 (DNA2.0 Inc., USA) is an S. cerevisiae expression vector containing a URA3 marker and a 2 um Ori sequence. FIG. 1 shows a cleavage map of the pJ1214-mhpF vector. In FIG. 1, P TEF indicates the TEF promoter. A nucleotide sequence of the pJ1214-mhpF vector is represented by SEQ ID NO. 59.
The MhpF gene of pJ1214-mhpF and ‘HIS3 cassette’ are linked to ‘pUC19 vector’ (NEB, N3041) using a restriction enzyme SalI, so as to prepare a pUC19-His-MhpF vector (SEQ ID NO. 60). The HIS3 cassette is an amplification product which is obtained by PCR using pRS413 (ATCC8758) as a template and primers of SEQ ID NO. 61 and SEQ ID NO. 62. mhpF is expressed in the presence of GPD promoter from the pUC19-His-MhpF vector.
PCR is performed using the prepared pUC19-His-MhpF vector as a template and primers of SEQ ID NOs. 63 and 64 in which a leu2 homologous recombination sequence and a promoter are linked, so as to prepare a mhpF insertion cassette. Here, the leu2 homologous recombination sequence does not express a functional protein due to its mutation in the parent strain.
The mhpF insertion cassette thus prepared is introduced into S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh). Introduction is performed by general heat shock transformation, and after transformation, cells are cultured in a histidine drop out medium (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, and Yeast synthetic drop-out without histidine (Sigma-Aldrich: Cat. no. Y1751) 1.9 g/L, glucose 2 (w/v) %) to replace the chromosomal Leu2 ORF with the cassette.
In order to examine introduction of the mhpF gene into the Leu2 locus in the strain thus obtained, PCR is performed using a genome of the cell as a template and a primer set of SEQ ID NOs. 65 and 66 as primers to examine gene deletion and gene introduction. As a result, S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, mhpF) is identified.
(3.2) Construction and Introduction of Vector for ald6 Deletion
An acetaldehyde dehydrogenase 6 (ald6) gene deletion cassette is amplified by PCR using the deletion vector pUC57-ura3HA as a template and a primer set of SEQ ID NOs. 67 and 68 as primers. The sequences of SEQ ID NOs. 67 and 68 include a region which is replaced for the ald6 gene by recombination with a homologous sequence in a chromosome of S. cerevisiae. 
In order to delete the ald6 gene from S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, mhpF) strain, the prepared “ald6 deletion cassette” is introduced into the strain by heat shock transformation. After heat shock, cells are cultured in a minimal Ura-drop out medium as a selection marker at 30° C. for 3 days to delete the chromosomal ald6 gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 69 and 70 to examine deletion of the ald6 gene.
As a result, S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, mhpF, Δald6) strain is identified.
(3.3) Construction and Introduction of Vector for EutE Introduction
(3.3.1) Construction of Yeast Dual Function Overexpression Vector, pCS-Ex1
PCR is performed using a pRS426GPD vector which is widely used as a yeast overexpression vector and a primer set of SEQ ID NO. 71 and SEQ ID NO. 72 to obtain a DNA fragment of 689 bp (GPD promoter). This DNA fragment is cloned into a KpnI-treated pCtB1 vector (Genbank Accession Number KJ922019) using an In-fusion kit (Clonetech, cat. 639650), and introduced into an E. coli cloning strain, TOP10 strain (Invitrogen, cat. C4040-06) by a general method. After introduction, the strain is plated on an LB agar plate (Bacto Tryptone 10 g/L, Yeast Extract 5 g/L, NaCl 10 g/L, and Bacto Agar 15 g/L) containing 50 ug/ml of kanamycin, followed by incubation. From colonies formed, plasmid DNAs are isolated, and plasmids having the same sequence as SEQ ID NO. 73 are examined. As a result, a yeast dual function overexpression vector, pCS-Ex1 is identified. Here, the dual function includes a gene expression after genomic integration of a gene and a gene expression on a vector.
(3.3.2) Construction of Yeast Dual Function E. coli eutE Gene Overexpression Vector
PCR is performed using genomic DNA of E. coli MG1655 strain and a primer combination of SEQ ID NO. 74 and SEQ ID NO. 75 so as to obtain a DNA fragment of 1447 bp, that is, EutE gene. This DNA fragment is mixed with a pCS-Ex1 vector which is treated with KpnI and SacI, and cloning is performed using an In-fusion kit (Clonetech cat. 639650), and introduced into an E. coli cloning strain, TOP10 strain (Invitrogen, cat. C4040-06) by a general method. After introduction, the strain is plated on an LB agar plate containing 50 ug/ml of kanamycin, followed by incubation. From colonies formed, plasmid DNAs are isolated, and plasmids having the same sequence as SEQ ID NO. 76 are examined. As a result, a yeast dual function overexpression vector, MD1040 is identified.
(3.3.3) Preparation of E. coli eutE Gene-Overexpressing Yeast
From the prepared MD1040 vector, a DNA fragment of 3985 bp is obtained by PCR using a primer combination of SEQ ID NO. 77 and SEQ ID NO. 78. This fragment is introduced into S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, mhpF, Δald6) by a general method, and then plated on a minimal medium, SD-URA agar plate containing no uracil (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, Yeast synthetic drop-out without uracil (Sigma-Aldrich: Cat. no. Y1501) 1.9 g/L, D-glucose 20 g/L, and Bacto Agar 20 g/L). After 3 days, from colonies formed, colonies which are confirmed to have a DNA fragment of 4357 bp by PCR using a primer combination of SEQ ID NO. 79 and SEQ ID NO. 80 are selected. From genomic DNA of a native strain, a DNA fragment of 2300 bp is obtained by PCR using a primer combination of SEQ ID NO. 79 and SEQ ID NO. 80. The obtained clones are inoculated in an YPD medium (Bacto Peptone 20 g/L, Yeast Extract 10 g/L, and D-glucose 20 g/L), and cultured at 30° C. under shaking at 230 rpm, and then plated on a counter-selection medium containing 5-FOA (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, Yeast synthetic drop-out without uracil (Sigma-Aldrich: Cat. no. Y1501) 1.9 g/L, Uracil 0.1 g/L, D-glucose 20 g/L, 5-fluoroorotic acid (5-FOA) 1 g/L, and Bacto Agar 20 g/L). After 3 days, from colonies formed, colonies which are confirmed to have a DNA fragment of 2963 bp by PCR using a primer combination of SEQ ID NO. C3 and SEQ ID NO. C4 are selected. As a result, S. cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, mhpF, Δald6, EutE) (hereinafter, referred to as “SP1130”) is identified.
(4) Preparation of S. cerevisiae CEN.PK2-1D SP1130 (ΔRGT1)
(4.1) Construction and Introduction of Vector for RGT1 Gene Deletion
A RGT1 gene deletion cassette is prepared by PCR using the above described deletion vector pUC57-Ura3HA as a template and a primer set of SEQ ID NO. 81 and 82 as primers. The sequences of SEQ ID NOs. 81 and 82 contain a region which is substituted for the RGT1 gene by recombination with a homologous sequence in a chromosome of S. cerevisiae. 
To delete the RGT1 gene from the S. cerevisiae CEN.PK2-1D SP1130 strain prepared in (3) of Example 1, the prepared “RGT1 gene deletion cassette” is introduced into the strain by heat shock transformation. After heat shock, the cells are cultured in a minimal Ura-drop out medium as a selection marker at 30° C. for 3 days to delete the chromosomal RGT1 gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 83 and 84 to examine deletion of the RGT1 gene.
As a result, S. cerevisiae CEN.PK2-1D SP1130 (ΔRGT1) strain is identified.
(5) Preparation of S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2ΔRGT1)
(5.1) Preparation of Pcyc1 Fragment and Construction of Recombinant Vector
Further, in order to obtain a DNA fragment containing a CYC1 promoter (Pcyc1) (SEQ ID NO. 85), a chromosomal DNA (gDNA) of the wild-type Saccharomyces cerevisiae strain CEN.PK2-1D is extracted using a Genomic-tip system of Qiagen (Company), and polymerase chain reaction is performed using the gDNA as a template and a PCR HL premix kit (manufactured by BIONEER, hereinafter, the same). For Pcyc1 amplification, PCR is performed using primers of SEQ ID NOs. 86 and 87 for 30 cycles consisting of denaturation at 94° C. for 30 seconds, annealing at 52° C. for 30 seconds, and elongation at 72° C. for 30 seconds. A PCR product is cleaved with EcoRI and a DNA fragment thus obtained (hereinafter, referred to as “Pcyc1 cassette”) is electrophoresed on a 0.8% agarose gel, followed by elution.
The pUC19-Trp1 vector (Appl Environ Microbiol. 2002 May; 68(5):2095-100) plasmid and the obtained Pcyc1 cassette are treated with the restriction enzyme EcoRI, respectively and ligated with each other to prepare a pUC19-Trp1-Pcyc1 vector. FIG. 3 shows the pUC19-Trp1-Pcyc1 vector.
(5.2) Preparation of S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2)
To replace PHXK2 with Pcyc1, the Pcyc1 cassette prepared in 5.1 of Example 1 is amplified to contain the HXT gene promoter sequence using primers of SEQ ID NOs. 88 and 89, mixed with 50% polyethylene glycol and a single stranded carrier DNA, and then incubated in a water bath at about 42° C. for about 30 minutes. The culture of Saccharomyces cerevisiae CEN.PK2-1D SP1130 is plated on a minimal solid medium containing no tryptophan (YSD, 6.7 g/L yeast nitrogen base without amino acids, 1.4 g/L Amino acid dropout mix (−trp)) and cultured at 30° C. for 3 days or longer. 10 colonies (mutant strain) are selected from colonies formed on the plate, and transferred onto a minimal solid medium containing no tryptophan, and also in a liquid medium having the same composition. Genomic DNA is isolated from the cultured strain using a commercial kit (Gentra Puregene Cell kit, Qiagen, USA). PCR is performed using the isolated genomic DNA of the mutant strain as a template and primers of SEQ ID NOs. 90 and 91 to confirm replacement of PHXK2 with Pcyc1. Then, a PCR product is subjected to electrophoresis to examine replacement of PHXK2 with Pcyc1. As a result, S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2) is identified.
(5.3) Preparation of S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2ΔRGT1)
To delete the RGT1 gene from the S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2) prepared in (5.2) of Example 1, the “RGT1 gene deletion cassette” prepared in (4.1) of Example 1 is introduced into the strain by heat shock transformation. After heat shock, the cells are cultured in a minimal Ura-drop out medium as a selection marker at 30° C. for 3 days to delete the chromosomal RGT1 gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 83 and 84 to examine deletion of the RGT1 gene.
As a result, S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2ΔRGT1) strain is identified.
(6) Preparation of S. cerevisiae CEN.PK2-1D SP1130 (ΔHXK2ΔRGT1)
(6.1) Construction and Introduction of Vector for HXK2 Gene Deletion
A HXK2 gene deletion cassette is prepared by PCR using the above described deletion vector pUC57-Ura3HA as a template and a primer set of SEQ ID NO. 92 and 93 as primers. The sequences of SEQ ID NOs. 92 and 93 contain a region which is substituted for the RGT1 gene by recombination with a homologous sequence in a chromosome of S. cerevisiae. 
To delete the HXK2 gene from the S. cerevisiae CEN.PK2-1D SP1130 strain prepared in (3) of Example 1, the prepared “HXK2 gene deletion cassette” is introduced into the strain by heat shock transformation. After heat shock, the cells are cultured in a minimal Ura-drop out medium as a selection marker at 30° C. for 3 days to delete the chromosomal HXK2 gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 94 and 95 to examine deletion of the HXK2 gene.
As a result, S. cerevisiae CEN.PK2-1D SP1130 (HXK2) strain is identified.
(6.2) Preparation of S. cerevisiae CEN.PK2-1D SP1130 (ΔRGT1ΔHXK2)
To delete the RGT1 gene from the S. cerevisiae CEN.PK2-1D SP1130 (ΔHXK2) strain prepared in (6.1) of Example 1, the “RGT1 gene deletion cassette” prepared in (4.1) of Example 1 is introduced into the strain by heat shock transformation. After heat shock, the cells are cultured in a minimal Ura-drop out medium as a selection marker at 30° C. for 3 days to delete the chromosomal RGT1 gene. For genotyping of the prepared strain, PCR is performed using a genome of the prepared strain as a template and a primer set of SEQ ID NOs. 83 and 84 to examine deletion of the RGT1 gene.
2. Test of Growth, Glucose Consumption, and Lactate Productivity of Prepared Yeast Cell
The prepared S. cerevisiae CEN.PK2-1D SP1130 (Δrgt1) and SP1130 (Pcyc1HXK2Δrgt1) strains are plated on an YPD solid medium (Yeast extract 1 (w/v) %, peptone 1 (w/v) %, and glucose 2 (w/v) %), respectively and cultured at 30° C. for 24 hours or longer. Then, the respective strains are inoculated in 25 ml of an YPD liquid medium containing 20 g/l of glucose in a 125 ml-flask, and cultured under aerobic conditions at 30° C. for 16 hours. The microaerobic conditions mean that the flask is sealed with a septum which does not allow ventilation, and agitated at 90 rpm. The control experiment is performed in the same manner, except that S. cerevisiae CEN.PK2-1D SP1130 strain is used.
Cell density in the culture is determined by measuring OD600 value using a spectrophotometer, and the culture is centrifuged to discard a supernatant, and a cell pellet is inoculated in a fresh YPD liquid medium containing 60 g/l of glucose in a 125 ml-flask to OD600 of 0.5. The flask is placed in a shaking incubator and the culture is incubated at 36° C. for 24 hours or longer under agitation at 90 rpm. The flask is sealed with a septum which does not allow ventilation.
During cultivation, samples are collected periodically, and centrifuged at about 13,000 rpm for about 10 minutes. Concentrations of various metabolites, lactate and glucose in a supernatant are analyzed by high-performance liquid chromatography (HPLC). The culture supernatant is filtered using a 0.45 um syringe filter and then L-lactate and glucose are quantified using an HPLC machine (Waters e2695 Separation Module instrument equipped with a Waters 2414 Differential Refractometer and a Waters 2998 Photodiode Array Detector (Waters, Milford, Mass.)). As an HPLC column, Aminex HPX-87H Organic Acid Analysis Column (300 mm×7.8 mm; Bio-Rad) that is equilibrated with 2.5 mM H2SO4 in water at 60° C. and at a flow rate of 0.5 mL/min is used.
Table 1 shows cell density, and glucose and lactate concentrations in the medium, which are measured after cultivation in the YPD medium under microaerobic conditions for 48 hours.











TABLE 1







Glucose
Lactate
Ethanol




consump-
produc-
produc-



Cell growth
tion
tivity
tivity


Strain
(max OD600)
(g/L)
(g/L)
(g/L)







SP1130
2.26
28.63
22.29
0.86


SP1130(Pcyc1HXK2)
2.62
31.52
24.75
1.18


SP1130(Δ rgt1)
2.46
36.16
27.06
0.99


SP1130(Pcyc1HXK2 Δ rgt1)
2.75
38.00
29.86
0.79


SP1130(Δ HXK2)
2.77
32.56
25.12
1.21


SP1130(Δ HXK2 Δ rgt1)
2.91
39.12
29.45
1.10









As in Table 1, SP1130 (Δrgt1) and SP1130 (Pcyc1HXK2Δrgt1) strains show 26% and 21% increases in glucose consumption and 21% and 21% increases in lactate production, compared to the control SP1130 and SP1130 (Pcyc1HXK2) strains, respectively. Therefore, when RGT1 gene is disrupted or activities of RGT1 gene and HXK2 gene are decreased, S. cerevisiae shows an unexpected increase in glucose consumption and lactate productivity.
3. Analysis of RNA Expression Levels of HXT Genes in Cells Having Decreased RGT1 and HXT2 Activities
Expression levels of HXT1, HXT2, HXT3, HXT4, and HXT7 genes in S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2ΔRGT1) strain are examined by qRT-PCT. Comparison of relative expression levels is performed by ΔΔCT, and TAF10 is used as a reference gene. The corresponding analysis is repeated three times. In detail, S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2ΔRGT1) strain is cultured in a 125 ml-flask, and sampling is performed at 24 hours, 32 hours, and 48 hours after the culture. Total RNAs are extracted using an RNeasy mini kit (Qiagen co.). The extracted RNA is used as a template to synthesize cDNA using a SuperScript® III First-Strand Synthesis System (Invitrogen). Then, primers that specifically bind to respective HXT genes, specifically, forward and reverse primers (SEQ ID NOs. 96 through 105, respectively) that specifically bind to HXT1, HXT2, HXT3, HXT4, and HXT7 genes are mixed with IQ SYBR Green Supermix (Bio-rad) in accordance with the manufacturer's protocol, respectively and their relative expression levels are examined using a qRT-PCR detection system (CFX96 realtime PCR detection system, Bio-rad).
Table 2 shows relative RNA expression levels of HXT1, HXT2, HXT3, HXT4, and HXT7 in S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2ΔRGT1) strain and S. cerevisiae CEN.PK2-1D SP1130 (Pcyc1HXK2) strain.











TABLE 2







24 hrs
36 hrs
48 hrs


Type of

after
after
after


RNA
Strain
culture
culture
culture



















HXT1
SP1130 (Pcyc1HXK2)
16.41
1.00
2.30



SP1130 (Pcyc1HXK2 Δ RGT1)
37.82
12.20
2.41


HXT2
SP1130 (Pcyc1HXK2)
1.03
1.00
1.02



SP1130 (Pcyc1HXK2 Δ RGT1)
57.43
49.25
5.73


HXT3
SP1130 (Pcyc1HXK2)
1.00
4.67
2.20



SP1130 (Pcyc1HXK2 Δ RGT1)
5.03
18.30
21.74


HXT4
SP1130 (Pcyc1HXK2)
0.00
1.00
0.00



SP1130 (Pcyc1HXK2 Δ RGT1)
8.09
0.88
50.88


HXT7
SP1130 (Pcyc1HXK2)
1.00
1.26
0.00



SP1130 (Pcyc1HXK2 Δ RGT1)
7.46
3.24
1.99









As shown in Table 2, a high expression level of HXT1 is observed at an early stage of culture in the strain having the decreased RGT1 activity (SP1130 (Pcyc1HXK2ΔRGT1)), compared to its parent cell (SP1130 (Pcyc1HXK2)). Further, a transient high expression of HXT2 is observed at a lag phase in the strain having the decreased RGT1 activity, compared to its parent cell. A high expression of HXT3 is observed at a growth arrest in the strain having the decreased RGT1 activity, compared to its parent cell. Furthermore, a high expression of HXT4 is observed at a growth phase in the strain having the decreased RGT1 activity, compared to its parent cell. A high expression of HXT7 is observed at a stationary phase in the strain having the decreased RGT1 activity, compared to its parent cell. These results suggest that the strain having the decreased RGT1 activity increases the HXT expression levels, compared to its parent cell.
It should be understood that the exemplary embodiments described herein 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.
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 comprising a genetic modification that decreases Restores Glucose Transport Protein 1 (RGT1) activity compared to the RGT1 protein activity of a parent cell thereof, wherein the genetically engineered yeast cell has increased productivity of a glycolytic intermediate compared to a parent cell thereof wherein the genetic modification is a disruption mutation of a gene encoding RGT1.

2. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell further comprises a genetic modification that decreases Hexokinase isoenzyme 2 (HXK2) protein activity compared to the HXK2 protein activity of a parent cell thereof.

3. The genetically engineered yeast cell of claim 2, wherein the genetic modification that decreases the HXK2 protein activity is a disruption mutation of a gene encoding HXK2.

4. The genetically engineered yeast cell of claim 1, wherein the yeast cell consumes glucose at an increased glucose consumption rate compared to the parent cell.

5. The genetically engineered yeast cell of claim 1, wherein the glycolytic intermediate is dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), pyruvate, or a combination thereof.

6. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell has increased productivity of glycerol-3-phosphate (G3P), glycerol, acetyl-CoA, ethanol, acetic acid, lactate, a TCA cycle intermediate, or a combination thereof.

7. The genetically engineered yeast cell of claim 6, wherein the genetically engineered yeast cell has increased productivity of a TCA cycle intermediate comprising citric acid, itaconic acid, isocitrate, oxalosuccinate, alpha-ketoglutarate, succinic acid, succinyl-CoA, fumaric acid, malate, oxaloacetate, or a combination thereof, and the pyruvate-derived substance is a product derived from the TCA cycle intermediate and the product is 1,3-butanediol (1,3-BDO), 1,4-butanediol (1,4-BDO), butanol, isobutanol, or a combination thereof.

8. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell further comprises an exogenous gene that encodes an enzyme that catalyzes conversion of pyruvate to lactate.

9. The genetically engineered yeast cell of claim 1, wherein the RGT1 protein has an amino acid sequence of SEQ ID NO. 1.

10. The genetically engineered yeast cell of claim 2, wherein the HXK2 protein has an amino acid sequence of SEQ ID NO. 3.

11. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell is a strain belonging to Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Shizosaccharomyces, Issachenkia, or Hansenula.

12. The genetically engineered yeast cell of claim 1, wherein the genetically engineered yeast cell further comprises a genetic modification that decreases the activity of an enzyme that catalyzes conversion of acetaldehyde to ethanol, an enzyme that catalyzes conversion of pyruvate to acetaldehyde, an enzyme that catalyzes conversion of lactate to pyruvate, an enzyme that catalyzes conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P), an enzyme that catalyzes conversion of glycerol-3-phosphate (G3P) to glycerol, an enzyme that catalyzes conversion of acetaldehyde to acetate, or a combination thereof.

13. The genetically engineered yeast cell of claim 12, wherein the enzyme that catalyzes conversion of acetaldehyde to ethanol belongs to EC 1.1.1.1, the enzyme that catalyzes conversion of pyruvate to acetaldehyde belongs to EC 4.1.1.1, the enzyme that catalyzes conversion of lactate to pyruvate belongs to EC 1.1.2.4 or EC 1.1.2.3, the enzyme that catalyzes conversion of DHAP to G3P belongs to EC 1.1.1.8, the enzyme that catalyzes conversion of glycerol-3-phosphate (G3P) to glycerol belongs to EC 3.1.3.21, and the enzyme that catalyzes conversion of acetaldehyde to acetate belongs to EC 1.2.1.4.

14. The genetically engineered yeast cell of claim 12, wherein the enzyme that catalyzes conversion of acetaldehyde to ethanol is alcohol dehydrogenase (ADH), the enzyme that catalyzes conversion of pyruvate to acetaldehyde is pyruvate decarboxylase (PDC), the enzyme that catalyzes conversion of lactate to pyruvate is lactate cytochrome-c oxidoreductase (CYB2), the enzyme that catalyzes conversion of DHAP to G3P is NAD-dependent glycerol-3-phosphate dehydrogenase (GPD), the enzyme that catalyzes conversion of glycerol-3-phosphate to glycerol is glycerol phosphate phosphatase (GPP), and the enzyme that catalyzes conversion of acetaldehyde to acetate is acetaldehyde dehydrogenase.

15. The genetically engineered yeast cell of claim 12, wherein a gene encoding the enzyme that catalyzes conversion of acetaldehyde to ethanol, a gene encoding the enzyme that catalyzes conversion of pyruvate to acetaldehyde, a gene encoding the enzyme that catalyzes conversion of lactate to pyruvate, a gene encoding the enzyme that catalyzes conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P), a gene encoding the enzyme that catalyzes conversion of glycerol-3-phosphate to glycerol, a gene encoding the enzyme that catalyzes conversion of acetaldehyde to acetate, or a combination thereof comprises a disruption mutation.

16. A method of producing a glycolytic intermediate or glycolytic intermediate-derived substance, the method comprising:
culturing the genetically engineered yeast cell of claim 1 in a cell culture medium to obtain a culture, whereby the genetically engineered yeast cell produces a glycolytic intermediate or glycolytic intermediate-derived substance in the culture; and
isolating the glycolytic intermediate from the culture.

17. The method of claim 16, wherein the culturing is performed under microaerobic conditions.

18. A method of producing a genetically engineered yeast cell having an ability to produce lactate, the method comprising:
introducing a gene encoding an enzyme that catalyzes the conversion of pyruvate into lactate into a yeast cell; and
disrupting a gene encoding an RGT1 protein in the yeast cell.

19. The method of claim 18, further comprising disrupting a gene encoding an HXK2 protein in the yeast cell.