# Patent

## Title
Genetically engineered and acid-resistant yeast cell with enhanced activity of radiation sensitivity complementing kinase and method of producing lactate by using the yeast cell

## Abstract

Provided is an acid-resistant yeast cell that is genetically engineered to enhance activity of a radiation sensitivity complementing kinase, and a method of producing lactate by using the yeast cell.

## Background
RELATED APPLICATION
This application claims the benefit of Korean Patent Application No. 10-2014-0094159, filed on Jul. 24, 2014, in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.
INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: One 164,396 byte ASCII (Text) file named “719887_ST25.TXT” created Jul. 21, 2015.
BACKGROUND
1. Field
The present disclosure relates to a genetically engineered and acid-resistant yeast cell with enhanced activity of radiation sensitivity complementing kinase and a method of producing lactate by using the yeast cell.
2. Description of the Related Art
Organic acids are widely used in a variety of industries. For example, lactate is an organic acid that is used in a variety of industrial fields, including food, pharmaceutical, chemical, and electronic industries. Lactate is a colorless, odorless, water-soluble, low-volatile material. Lactate is also not toxic to the human body, and is used as a flavoring agent, a sour taste agent, a preserving agent, or the like. Lactate is also used as a source of polylactic acid (PLA) that is an environmentally friendly, biodegradable plastic known as an alternate polymeric material.
Organic acids may be dissociated into hydrogen ions and their own negative ions at a higher pH than their own dissociation constant (pka value), for example, under a neutral condition. Meanwhile, organic acids, for example, lactic acid, may be present in the form of free acid without an electromagnetic force at a pH lower than its own pKa value. An organic acid in the form of negative ions may not be permeable with respect to a cell membrane, but may be permeable with respect to the cell membrane when it is present in the form of free acid. Therefore, an organic acid in free acid form may flow into the cells from extracellular environments where the concentration of the organic acid is high, and thus lower an intercellular pH level. Meanwhile, an organic acid present as negative ions requires an additional isolation process involving the addition of a salt. Also, a cell lacking acid-resistance may become inactive and die under acidic conditions, such as in the case of lactic acid.
Therefore, there is a need for a microorganism with acid-resistance.
SUMMARY
Provided is an acid-resistant yeast cell that is genetically engineered to have enhanced activity of radiation sensitivity complementary kinase. In some embodiments, the yeast cell comprises an exogenous polynucleotide encoding a radiation sensitivity complementing kinase; a heterologous promoter operatively linked to a polynucleotide encoding a radiation sensitivity complementing kinase, or both.
Also provided is a method of producing lactate by culturing the yeast cell.
Further provided is a method of increasing the acid resistance or lactate production of a yeast cell by increasing the expression of a radiation sensitivity complementing kinase in the yeast cell.
Additional aspects will be set forth in part in the description which follows and, in part, will be apparent from the description, or may be learned by practice of the presented embodiments.



BRIEF DESCRIPTION OF THE DRAWINGS
These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:
FIG. 1 is a vector map depicting a p416-CCW12p-LDH vector;
FIG. 2 is a vector map depicting a pUC19-HIS3 vector;
FIG. 3 is a vector map depicting a pUC19-CCW12p-LDH-HIS3 vector;
FIG. 4 is a vector map depicting a pUC19-Trp1-Pcyc1 vector;
FIG. 5 is a schematic illustrating a process of manufacturing a strain to which a RCK1 promoter substitution vector is inserted in a parent strain Saccharomyces cerevisiae; 
FIG. 6 is a vector map depicting a P57 vector;
FIG. 7 is a vector map depicting a p57_ura3HA-PTPI1 vector;
FIG. 8 is a vector map depicting a p416-PGK-RCK1 vector;
FIG. 9 is a graph that shows concentrations of lactic acid and glucose according to incubation in Control group 1 and Experiment group 1;
FIG. 10 is a graph that shows concentrations of lactic acid and glucose according to incubation in Control group 2 and Experiment group 2;
FIG. 11 is a graph that shows concentrations of lactic acid and glucose according to incubation in Control group 3 and Experiment group 3;
FIG. 12 is a graph that shows concentrations of lactic acid and glucose according to incubation in Control group 1 and Experiment group 1; and
FIG. 13 is a series of images depicting the results of yeast cells cultured in an acidic YPD medium containing 45 g/L of lactic acid and having a pH of 3.5.



DETAILED DESCRIPTION
Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.
As used herein, the term “activity increase” or “increased activity” of a cell, enzyme, polypeptide, or protein denotes that the activity level of a genetically modified (i.e., genetically engineered) cell or an enzyme or a polypeptide produced by a genetically modified cell may refer to an increase of activity of an enzyme, a polypeptide, or a protein sufficient enough to show activity and mean that the activity level of a cell or an isolated polypeptide is higher than an activity level measured in the same kind of comparable cell, parent cell, or the original polypeptide. The activity of a subject genetically engineered cell or polypeptide (or enzyme or protein) may be increased by any amount, such as 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 about 200% or about 300% or more compared to the activity of the original non-engineered cell or polypeptide, such as the parent cell of a genetically engineered cell, or a polypeptide of the parent cell, or the wild-type cell or polypeptide (or enzyme or protein). Enhanced activity may be confirmed by using any method commonly known in the art.
Increased activity of a polypeptide (e.g., protein, or enzyme) may result from an increase in polypeptide expression level or increased specific activity of the polypeptide. The increased specific activity of the polypeptide may be due to enzyme engineering, such as by mutation or random mutagenesis of one or more specific amino acids of an active domain in the enzyme or polypeptide. The expression level increase may be due to the introduction of a polynucleotide encoding a polypeptide into a cell, by the increased number of copies of the polynucleotide in the cell, or by mutation of a regulatory region of the polynucleotide. The mutation of a regulatory region of the polynucleotide may include modification of an expression regulatory sequence of a gene. The (expression) regulatory sequence may be a promoter sequence for expression of the gene or a transcription terminator sequence. Also, the (expression) regulatory sequence may be a sequence encoding a motif that may affect the gene expression. Examples of the motif may include 2D stabilizing motifs, RNA instability motifs, splice-activating motifs, polyadenylation motifs, adenine-rich sequences, and endonuclease recognition sites.
Here, the polynucleotide introduced from the outside of the cell or the polynucleotide having increased copy number may be an endogenous gene or an exogenous gene. The endogenous gene may refer to a gene present in a genetic material contained within the microorganism. The exogenous gene may refer to a gene that is introduced to a host cell, such as a gene integrated into a host cell genome. The exogenous gene introduced into a host cell may be a homologous or heterologous with respect to the host cell. The term “heterologous” denotes that the gene is a foreign gene and is non-native to the cell into which it is inserted.
The expression “increased copy number” may include a case where a copy number increase occurs by an introduction of an exogenous gene or amplification of an endogenous gene. The introduction of the gene may occur by using a vehicle such as a vector. The introduction may be a transient introduction, in which the gene is not integrated into the genome, or integration into the genome. The introduction may, for example, occur by introducing a vector inserted with a polynucleotide encoding a desired polypeptide into the cell and then replicating the vector in the cell or integrating the polynucleotide into the genome of the cell and then replicating the polynucleotide together with the replication of the genome.
As used herein, the term “gene” denotes a polynucleotide that encodes a product and that can be expressed by at least one of transcription and translation, where an example of the product is mRNA or an encoded protein. A gene may include a coding region and, optionally, a regulatory sequence such as a 5′-non coding sequence and a 3′-non coding sequence in addition to the coding region.
The terms “cell”, “strain”, or “microorganism” as used herein may be interchangeably used and may include bacteria, yeast, or fungi.
As used herein, the term “activity reduction” or “decreased activity” of an enzyme or a polypeptide denotes a cell, an isolated enzyme, or a polypeptide whose activity is lower than activity measured in a comparable cell of the same type, such as a parent cell of an engineered cell, or the original “wild type” non-engineered polypeptide, enzyme or cell. Reduced and decreased activity encompasses no activity. The activity of a subject genetically engineered polypeptide, enzyme, or cell may be decreased by any amount, such as about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% in comparison to the same biochemical activity of a polypeptide, enzyme, or cell that is not genetically engineered, such as a wild-type polypeptide, enzyme, or cell, or the activity of the parent cell of a genetically engineered cell. The decreased enzyme activity may be confirmed by using any commonly known method in the art. The decrease in activity may include the case of an enzyme having no activity or reduced activity even when the enzyme is expressed, a gene encoding the enzyme that is not expressed, or a decrease in an expression amount of the gene compared to that of a gene encoding an originally not engineered polypeptide or a wild-type polypeptide even when the gene encoding the enzyme is expressed.
Activity of the enzyme may be reduced due to deletion or disruption of a gene that encodes the enzyme. As used herein, the “deletion” or “disruption” of the gene includes mutation (insertion, deletion, or substitution) or deletion of all or part of a gene or regulatory region (e.g., promoter or terminator of a gene) such that the gene is not expressed or has a reduced amount of expression, or the activity of the gene product (e.g., enzyme) is removed or reduced even when the gene is expressed. The deletion or disruption of the gene may be caused by genetic engineering such as homologous recombination, mutation induction, or molecular evolution. When a cell includes a plurality of the same genes or at least two different polypeptide paralogs, at least one gene may be deleted or disrupted.
As used herein, the term “sequence identity” of a polypeptide or polynucleotide with respect to another polypeptide or polynucleotide refers to a degree of sameness in an amino acid sequence or a nucleic acid sequence in a specific region of two sequences that are aligned to best match each other for comparison. The sequence identity is a value obtained via optimal alignment and comparison of the two sequences in the specific region for comparison, in which a partial sequence in the specific region for comparison may be added or deleted with respect to a reference sequence. The sequence identity represented in a percentage may be calculated by, for example, comparing two sequences that are aligned to best match each other in the specific region for comparison, determining matched sites with the same amino acid or base in the two sequences to obtain the number of the matched sites, dividing the number of the matched sites in the two sequences by a total number of sites in the compared specific regions (i.e., a size of the compared region), and multiplying a result of the division by 100 to obtain a sequence identity as a percentage. The sequence identity as a percentage may be determined using a known sequence comparison program, for example, BLASTP or BLASTN (NCBI), CLC Main Workbench (CLC bio), or MegAlignTM (DNASTAR Inc).
In identifying a polypeptide or polynucleotide with the same or similar function or activity with respect to various types of species, a suitable levels of sequence identity may be applied. In some embodiments, the sequence identity may be, for example, about 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 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% or more.
As used herein, the term “parent cell” may denote a cell without specific genetic modification that produces a genetically engineered cell. Also, the term “wild-type” polypeptide or polynucleotide may denote a polypeptide or a polynucleotide without specific genetic modification, where the specific genetic modification may result in a genetically engineered polypeptide or polynucleotide. For instance, with regard to a cell that is engineered to increase activity of a radiation sensitivity complementing kinase, a parent cell may be a cell of the same type that is not genetically engineered to increase activity of radiation sensitivity complementing kinase. The parent cell may be a parent strain that is genetically modified to increase activity of radiation sensitivity complementing kinase, thereby providing the genetically engineered cell. As used herein, the term “lactate” includes its anion form, a salt thereof, a solvate, a polymorph, or a combination thereof in addition to lactic acid itself. The salt may be, for example, an inorganic acid salt, an organic acid salt, or a metal salt. The inorganic acid salt may be a hydrochloride, bromate, phosphate, sulfate, or disulfate. The inorganic acid salt may be formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edisylate, trifluoroacetate, benzoate, gluconate, methansulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, or aspartate. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.
According to an exemplary embodiment, an acid-resistant yeast cell has increased activity of radiation sensitivity complementing kinase.
The radiation sensitivity complementing kinase (RCK) may be serine/threonine-protein kinase, which is an enzyme classified under EC 2.7.11.1. The radiation sensitivity complementing kinase may be RCK1 or RCK2. The radiation sensitivity complementing kinase may include an amino acid sequence having about 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, RCK1 and RCK2 may each have an amino acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2. The radiation sensitivity complementing kinase may be a polynucleotide sequence that encodes a protein having at least 95% sequence identity with SEQ ID NO: 1 or 2, or may be a polynucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In some other embodiments, rck1 and rck2 genes may each have a polynucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4.
The yeast cell with an acid-resistant property may have an increased growth rate under an acid condition compared to the growth rate of a parent cell. The acid condition may be an acid condition including an organic acid, an inorganic acid, or a combination thereof. The organic acid may be an organic acid having 1 to 20 carbon atoms. The organic acid may be acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, 4-hydroxybutyric acid, succinic acid, fumaric acid, malic acid, oxalic acid, adipic acid, or a combination thereof. The yeast cell may have an increased growth rate as compared to a parent cell under a pH condition in a range of about 2.0 to about 7.0, for example, pH in a range of about 2.0 to about 5.0, about 2.0 to about 4.5, about 2.0 to about 4.0, about 2.0 to about 3.8, about 2.5 to about 3.8, about 3.0 to about 3.8, about 2.0 to about 3.0, about 2.0 to about 2.7, about 2.0 to about 2.5, or about 2.5 to about 3.0 compared to that of a yeast cell in which activity of the radiation sensitivity complementing kinase is not increased.
Also, the yeast cell with an acid-resistant property may have a higher survival rate under an acid condition compared to that of a parent cell. The acid condition may be an acid condition including an organic acid, an inorganic acid, or a combination thereof. The organic acid may be an organic acid having 1 to 20 carbon atoms. The organic acid may be acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, 4-hydroxybutyric acid, succinic acid, fumaric acid, malic acid, oxalic acid, adipic acid, or a combination thereof. The yeast cell may have a higher survival rate under a pH condition in a range of about 2.0 to about 7.0, for example, pH in a range of about 2.0 to about 5.0, about 2.0 to about 4.5, about 2.0 to about 4.0, about 2.0 to about 3.8, about 2.5 to about 3.8, about 3.0 to about 3.8, about 2.0 to about 3.0, about 2.0 to about 2.7, about 2.0 to about 2.5, or about 2.5 to about 3.0 compared to that of a yeast cell in which activity of the radiation sensitivity complementing kinase is not increased.
Also, the yeast cell with an acid-resistant property of may have increased metabolism under an acid condition compared to that of a parent cell. The acid condition may be an acid condition including an organic acid, an inorganic acid, or a combination thereof. The organic acid may be an organic acid having 1 to 20 carbon atoms. The organic acid may be acetic acid, lactic acid, propionic acid, 3-hydroxypropionic acid, butyric acid, 4-hydroxybutyric acid, succinic acid, fumaric acid, malic acid, oxalic acid, adipic acid, or a combination thereof. The yeast cell may have increased metabolism under a pH condition in a range of about 2.0 to about 7.0, for example, pH in a range of about 2.0 to about 5.0, about 2.0 to about 4.5, about 2.0 to about 4.0, about 2.0 to about 3.8, about 2.5 to about 3.8, about 3.0 to about 3.8, about 2.0 to about 3.0, about 2.0 to about 2.7, about 2.0 to about 2.5, or about 2.5 to about 3.0 compared to that of a yeast cell in which activity of the radiation sensitivity complementing kinase is not increased. Here, a degree of “metabolism” may be measured by a nutrition uptake rate per cell, for example, a glucose uptake rate per cell. Also, a degree of “metabolism” may be measured by a product secretion rate per cell, for example, a carbon dioxide secretion rate per cell.
The yeast cell may belong to Saccharomyces genus, Kluyveromyces genus, Candida genus, Pichia genus, Issatchenkia genus, Debaryomyces genus, Zygosaccharomyces genus, Shizosaccharomyces genus, or Saccharomycopsis genus. Saccharomyces genus may be, for example, S. cerevisiae, S. bayanus, S. boulardii, S. bulderi, S. cariocanus, S. cariocus, S. chevalieri, S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, S. florentinus, S. kluyveri, S. martiniae, S. monacensis, S. norbensis, S. paradoxus, S. pastorianus, S. spencerorum, S. turicensis, S. unisporus, S. uvarum, or S. zonatus. 
The increase in activity of RCK may be due to the increased copy number of the gene, or due to modification of an expression regulatory sequence (e.g., promoter) of the gene. The increased copy number may be caused by introduction of a gene from outside to inside of the cell or by amplification of an endogenous gene.
The introduction of a gene may be performed by mediation of vehicles, such as a vector. The introduction may be transient introduction that is not integrated to a genome or introduction inserted in a genome. For example, the introduction may be performed by introducing a vector, to which the gene is inserted, to a cell, and copying the vector in the cell or integrating the gene into the genome. The gene may be operably connected to a regulatory sequence involved in regulation of expression of the gene. The regulatory sequence may include a promoter, a 5′-non coding sequence, a 3′-non coding sequence, an transcription terminator sequence, an enhance, or a combination thereof. The gene may be an endogenous gene or an exogenous gene. Also, the regulatory sequence may be a sequence encoding a motif that may affect the gene expression. Examples of the motif may include 2D stabilizing motifs, RNA instability motifs, splice-activating motifs, polyadenylation motifs, adenine-rich sequences, and endonuclease recognition sites.
The increase in activity of RCK may be caused by mutation of the one or more genes encoding the RCK. The mutation may cause substitution, insertion, addition, or conversion of at least one base.
The increase in activity of RCK can be caused by linking an endogenous or exogenous gene to a promoter with increased activity as compared to the native RCK promoter of a given cell (e.g., a heterologous promoter that does not normally drive expression of RCK). Suitable promoters may include a constitutive promoter. The constitutive promoter may be derived from triose phosphate isomerase (TPI1), covalently linked cell wall protein 12 (CCW12), pyruvate decarboxylase 1 (PDC1), phosphoglycerate kinase (PGK1), transcription enhancer factor-1 (TEF-1), glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, TDH3, GPD), purine-cytosine permease (PCPL3), alcohol dehydrogenase (ADH1) genes, or combination thereof.
The yeast cell may have a capability of producing lactate. The yeast cell may have an activity of a polypeptide that converts pyruvate into lactate. The yeast cell may include a gene encoding a polypeptide that converts pyruvate into lactate. In the yeast cell, the activity of a polypeptide that converts pyruvate into lactate may be increased. The polypeptide that converts pyruvate into lactate may be a lactate dehydrogenase (LDH). The LDH may be an NAD(P)-dependent enzyme. Also, the LDH may be stereo-specific and may produce only L-lactate, only D-lactate, or both L-lactate and D-lactate. The NAD(P)-dependent enzyme may be an enzyme classified under EC 1.1.1.27 that converts pyruvate into L-lactate, or an enzyme classified under EC 1.1.1.28 that converts pyruvate into D-lactate.
In the yeast cell having a capability of producing lactate, an activity of LDH may be increased. The yeast cell may include a gene encoding at least one LDH, and the gene may be exogenous. A polynucleotide may be derived from bacteria, yeasts, fungi, mammals or reptiles. The polynucleotide may be a polynucleotide that encodes at least one LDH selected from the group consisting of Lactobacillus helveticus, L. bulgaricus, L. johnsonii, L. plantarum, Pelodiscus sinensis japonicus, Ornithorhynchus anatinus, Tursiops truncatus, Rattus norvegicus, Xenopus laevis, and Bos Taurus. An LDH derived from Pelodiscus sinensis japonicus, an LDH derived from Ornithorhynchus anatinus, an LDH derived from Tursiops truncatus, and an LDH derived from Rattus norvegicus may each include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with amino acids of SEQ ID NOS: 5, 6, 7, 8, and 9. In some embodiments, a polynucleotide encoding the LDH may be a polynucleotide that encodes an amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NOS: 5, 6, 7, 8, and 9. In some other embodiments, a polynucleotide encoding the LDH may include a polynucleotide sequence that encodes an amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NOS: 5, 6, 7, and 8, a polynucleotide sequence of SEQ ID NO: 10, or a polynucleotide sequence of SEQ ID NO: 11.
A polynucleotide encoding the LDH may be included in a vector. Examples of the vector may include a replication origin, a promoter, a LDH-encoding polynucleotide, and a terminator. The replication origin may include a yeast autonomous replication sequence (ARS). The yeast ARS may be stabilized by a yeast centrometric sequence (CEN). The promoter may be selected from the group consisting of a cytochrome c (CYC) promoter, a transcription elongation factor (TEF) promoter, a glycerol-3-phosphate dehydrogenase (GPD) promoter, an alcohol dehydrogenase (ADH) promoter, a promoter of CCW12 gene, and a promoter of PGK gene. The CYC promoter, the TEF promoter, the GPD promoter, the ADH promoter, the promoter of CCW12, and the promoter of PGK gene may each have a nucleotide sequence of SEQ ID NOS: 38, 39, 40, 41, 42, and 43. The terminator may be selected from the group consisting of a terminator of a gene encoding a phosphoglycerate kinase 1 (PGK1), a terminator of a gene encoding a cytochrome c 1 (CYC1), and a terminator of a gene encoding galactokinase 1 (GAL1). The CYC1 terminator may have a nucleotide sequence of SEQ ID NO: 44. The vector may further include a selection marker. The LDH-encoding polynucleotide may be included in a genome at a specific location of a yeast cell. The specific location of the yeast cell may include a locus of a gene to be deleted and disrupted, such as pyruvate decarboxylase (PDC) or cytochrome-c oxidoreductase 2 (CYB2). When the LDH-encoding polynucleotide functions to produce an active protein in a cell, the polynucleotide is considered as “functional” in a cell.
The yeast cell may include a polynucleotide that encodes one LDH or a polynucleotide that encodes multiple LDH copies, e.g., 2 to 10 copies. The yeast cell may include a polynucleotide that encodes multiple LDH copies into, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 copies. When the yeast cell includes the polynucleotide encoding multiple LDHs, each polynucleotide may include copies of the same LDH polynucleotide or copies of polynucleotides encoding at least two different LDHs. The multiple copies of the polynucleotide encoding exogenous LDHs may be included in the same locus or multiple loci in a genome of a host cell, and the promoter or terminator of each copy of the polynucleotide may be identical to or different from each other.
Also, the yeast cell may have a capability of producing lactate. Activity of the yeast cell interrupting flow of metabolic products for producing lactate may be inactivated or reduced. In the yeast cell, activity of a pathway catalyzing or assisting the flow of metabolic products for producing lactate may be increased.
In the yeast cell, activity of a polypeptide that converts pyruvate into acetaldehyde, a polypeptide that converts lactate into pyruvate, a polypeptide that converts dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate, a polynucleotide that encodes an external mitochondrial NADH dehydrogenase, a mitochondrial pyruvate carrier (MPC), a polypeptide that converts pyruvate into oxaloacetate, a polypeptide that converts acetaldehyde into ethanol, Fps1, or a combination thereof may be reduced.
In the yeast cell, a gene encoding the polypeptide that converts pyruvate into acetaldehyde may be deleted or disrupted. The polypeptide that converts pyruvate into acetaldehyde may be an enzyme that is classified under EC 4.1.1.1. The polypeptide that converts pyruvate to acetaldehyde may be a pyruvate decarboxylase, e.g., PDC1, PDC5 or PDC6. The polypeptide that converts pyruvate to acetaldehyde may include an amino acid sequence having a sequence identity of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more with an amino acid sequence of SEQ ID NO: 12. The gene encoding the polypeptide that converts pyruvate to acetaldehyde may be a polynucleotide that encodes an amino acid sequence having about 95% or more sequence identity with respect to an amino acid sequence of SEQ ID NO: 12, or may have a polynucleotide sequence of SEQ ID NO: 13. For example, the gene may be pdc1, pdc5 or pdc6.
In the yeast cell, a gene encoding the polypeptide that converts lactate into pyruvate may be deleted or disrupted. The polypeptide that converts lactate into pyruvate may be a CYC-dependent enzyme. The polypeptide that converts lactate into pyruvate may be an enzyme that is classified under EC 1.1.2.4 that acts on D-lactate or EC 1.1.2.3 that acts on L-lactate. The polypeptide that converts lactate into pyruvate may be lactate cytochrome c-oxidoreductase, for example, a CYB2 (CAA86721.1), a CYB2A, a CYB2B, a DLD1, a DLD2, or a DLD3. The polypeptide that converts lactate into pyruvate may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 12. The gene encoding the polypeptide that converts lactate into pyruvate may be a polynucleotide sequence that encodes an amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NO: 14, or may include a polynucleotide sequence of SEQ ID NO: 15.
In the yeast cell, a gene encoding the polypeptide that converts DHAP into glycerol-3-phosphate may be deleted or disrupted. The polypeptide that converts DHAP into glycerol-3-phosphate may be a cytosolic glycerol-3-phosphate dehydrogenase and may be an enzyme that catalyzes reduction of DHAP to glycerol-3-phosphate by using oxidation of NADP to NAD+ or NADP+. The polypeptide may be classified under EC 1.1.1.8. The cytosolic glycerol-3-phosphate dehydrogenase may be GPD1. The cytosolic glycerol-3-phosphate dehydrogenase may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 16. A gene encoding the cytosolic glycerol-3-phosphate dehydrogenase may include a polynucleotide sequence encoding an amino acid sequence having a sequence identity of about 95% or more with an amino acid sequence of SEQ ID NO: 14, or may include a polynucleotide sequence of SEQ ID NO: 15.
In the yeast cell, a polynucleotide that encodes the external mitochondrial NADH dehydrogenase may be deleted or disrupted. The external mitochondrial NADH dehydrogenase may be an enzyme classified under EC. 1.6.5.9 or EC. 1.6.5.3. The external mitochondrial NADH dehydrogenase may be a type II NADH: ubiquinone oxidoreductase. The external mitochondrial NADH dehydrogenase may be located on the outer surface of the inner mitochondrial facing a cytoplasm. The external mitochondrial NADH dehydrogenase may be an enzyme catalyzing oxidation of cytosolic NADH to NAD+. The external mitochondrial NADH dehydrogenase may re-oxidize cytosolic NADH formed by a glycolysis process. The external mitochondrial NADH dehydrogenase may provide cytosolic NADH to a mitochondrial respiratory chain. The external NADH dehydrogenase may be NDE1, NDE2, or a combination thereof. The external mitochondrial NADH dehydrogenase may be distinguished from an internal mitochondrial NADH dehydrogenase NDI1 that is present and functions inside mitochondria. The external mitochondrial NADH dehydrogenase may include an amino acid sequence having a sequence identity of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more with an amino acid sequence of SEQ ID NO: 18 or 19. For example, NDE1 and NDE2 may each have an amino acid sequence of SEQ ID NO: 18 and 19. The gene encoding the external mitochondrial NADH dehydrogenase may be a polynucleotide sequence encoding an amino acid sequence having a sequence identity of about 95% or more with an amino acid sequence of SEQ ID NO: 18 or 19, or may have a polynucleotide sequence of SEQ ID NO: 20 or 21. In some embodiments, the nde1 gene may include a polynucleotide sequence of SEQ ID NO: 20, and the nde2 gene may include a polynucleotide sequence of SEQ ID NO: 21.
In the yeast cell, a polynucleotide that encodes the mitochondrial pyruvate carrier (MPC) may be deleted or disrupted. The MPC may be a polypeptide that is present in a mitochondrial inner membrane and functions to move cytoplasmic pyruvate to the mitochondria. The MPC may be MPC 1, MPC2, MPC3, or a combination thereof. In some embodiments, the MPC may include an amino acid sequence having a sequence identity of about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more with an amino acid sequence of SEQ ID NO: 22, 23, or 24. In detail, the MPC may include an amino acid sequence having a sequence identity of about 95% or more with an amino acid sequence of SEQ ID NO: 22, 23, and 24. The MPC 1, the MPC2, and the MPC3 may each have an amino acid sequence of SEQ ID NOS: 22, 23, and 24. The polynucleotide encoding the MPC may be a polynucleotide encoding a protein having a sequence identity of about 95% or more with an amino acid sequence of SEQ ID NO: 22, 23, or 24. In some other embodiments, the MPC may include a polynucleotide sequence of SEQ ID NO: 25, 26, or 27. The sequences of SEQ ID NOS: 25, 26, and 27 are each a polynucleotide encoding the MPC 1, the MPC2, and the MPC3.
In the yeast cell, the activity of a polypeptide that converts pyruvate into oxaloacetate may be reduced. The polypeptide that converts pyruvate into oxaloacetate may catalyze Mg-ATP dependent carboxylation reaction and biotin-dependent carboxylation reaction of pyruvate to oxaloacetate. The polypeptide may convert pyruvate into oxaloacetate by consuming one molecule of ATP. The polypeptide may be an enzyme classified under EC 6.4.1.1. The polypeptide may be two isoenzymes of pyruvate carboxylase (Pyc), e.g., PYC1 and/or PYC2, in Saccharomyces genus. The polypeptide that converts pyruvate into oxaloacetate may have an amino acid sequence having about 50% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or 100% with an amino acid sequence of SEQ ID NO: 28 (GenBank ID: NP_011453.1) and/or SEQ ID NO: 29 (GenBank ID: NP_009777.1). The two isoenzymes of pyruvate carboxylase may be encoded by separate genes. These genes may each include a nucleotide sequence of SEQ ID NO: 30 (GI Number: 6321376) and/or SEQ ID NO: 31 (GI Number: 63196950). These genes may be pyc1 or pyc2 encoding a pyruvate carboxylase (PYC). In the yeast cell, a gene encoding the polypeptide that converts pyruvate into oxaloacetate may have modification or mutation of an expression regulatory sequence of the gene. For example, the modification or mutation of the expression regulatory sequence in genes that encode PYC1, PYC2, or a combination thereof may be performed by substitution with a promoter having lower expression level than that of a promoter of a parent cell, e.g., PPYC1 and PPYC2 Examples of the promoter having low expression level include a LEUM promoter (Pleum), a cyc1 promoter, (Pcyc1), or a mutant thereof. Meanwhile, examples of the promoter having lower expression level than that of the promoter of the parent cell, e.g., PPYC1 and PPYC2, may include a TEF1 promoter, a GPC promoter, or a GAL promoter.
In the yeast cell, a gene encoding the polypeptide that converts acetaldehyde into ethanol may be deleted or disrupted. The polypeptide may be an enzyme that catalyzes conversion of acetaldehyde to ethanol. The polypeptide may be classified under EC. 1.1.1.1. The polypeptide may be an enzyme that catalyzes conversion of acetaldehyde to ethanol. The polypeptide may be an alcohol dehydrogenase (Adh), for example, Adh 4. The polypeptide may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 32. The gene encoding the polypeptide may include a polynucleotide sequence encoding an amino acid sequence having about 95% or more of sequence identity with an amino acid sequence of SEQ ID NO: 32, or it may include a polynucleotide sequence of SEQ ID NO: 33. For example, the gene may be adh4.
In the yeast cell, a polynucleotide that encodes Fps1 may be deleted or disrupted. The Fps1 may be aquaglyceroporin, and may be referred to as glycerol channel protein, glycerol transport polypeptide, glycerol facilitator channel, or glycerol uptake/efflux facilitator protein. Glycerol may be secreted outside the cell by using the Fps1. The Fps1 may be classified as TCDB 1.A.8.5.1 in transporter classification system provided by Transport Classification Database (M. Saier; U of CA, San Diego). The Fps1 protein (Fps1p) may include an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 34. A polynucleotide encoding the Fps1p may have a polynucleotide sequence encoding amino acid sequence having about 95% or more sequence identity with an amino acid sequence of SEQ ID NO: 34, or it may have a polynucleotide sequence of SEQ ID NO: 35.
In the yeast cell, the activity of a plasma membrane transporter may be increased. The term “plasma membrane transporter” may refer to a polypeptide catalyzing organic products to move across the plasma membrane, and in some embodiments, may be a carboxylic acid transporter. The carboxylic acid transporter may be a monocarboxylate permease, and in some embodiments, may be Jen1. Jen1 may have an amino acid sequence having about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with an amino acid sequence of SEQ ID NO: 36. A polynucleotide encoding the Jen1 may be a polynucleotide encoding an amino acid sequence having about 95% sequence identity with an amino acid sequence of SEQ ID NO: 36, or may be a polynucleotide sequence of SEQ ID NO: 37.
Also, in the yeast cells according to an exemplary embodiment, activity of the radiation sensitivity complementing kinase is increased as compared with that of a parent cell; a gene that encodes a polypeptide converting pyruvate into acetaldehyde, a gene that encodes a polypeptide converting lactate to pyruvate, a gene that encodes a polypeptide converting dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate, or a combination thereof is deleted or disrupted; and a gene that encodes a polypeptide converting pyruvate into lactate is included or additionally introduced to the yeast cell. The yeast cell may be Saccharomyces cerevisiae. In some embodiments, the yeast cell in which a gene that encodes a polypeptide converting pyruvate into acetaldehyde, a gene that encodes a polypeptide converting lactate into pyruvate, a gene that encodes a polypeptide converting DHAP into glycerol-3-phosphate, or a combination thereof is deleted or disrupted; and to which a gene that encodes a polypeptide converting pyruvate into lactate is included or additionally introduced may be have accession number: KCTC 12415 BP.
In the yeast cells according to another exemplary embodiment, activity of the radiation sensitivity complementing kinase is increased as compared with that of a parent cell; a gene that encodes a polypeptide converting pyruvate into acetaldehyde, a gene that encodes a polypeptide converting lactate into pyruvate, a gene that encodes a polypeptide converting DHAP into glycerol-3-phosphate, a polynucleotide that encodes an external mitochondrial NADH dehydrogenase, an MPC, a polypeptide that converts pyruvate into oxaloacetate, a polypeptide that converts acetaldehyde into ethanol, or a combination thereof is deleted or disrupted; and a gene that encodes a polypeptide converting pyruvate into lactate is included or additionally introduced to the yeast cell. The yeast cell may be Saccharomyces cerevisiae. 
In the yeast cells according to another exemplary embodiment, activity of the radiation sensitivity complementing kinase is increased as compared with that of a parent cell; a gene that encodes a polypeptide converting pyruvate into acetaldehyde, a gene that encodes a polypeptide converting lactate into pyruvate, a gene that encodes a polypeptide converting DHAP into glycerol-3-phosphate, a polynucleotide that encodes an external mitochondrial NADH dehydrogenase, an MPC, a polypeptide that converts pyruvate into oxaloacetate, a polypeptide that converts acetaldehyde into ethanol, Fps1, or a combination thereof is deleted or disrupted; and a gene that encodes a polypeptide converting pyruvate into lactate is included or additionally introduced to the yeast cell. The yeast cell may be Saccharomyces cerevisiae. 
Also provided herein is a method for preparing a recombinant yeast cell having increased acid resistance and/or increased lactate production, and method for increasing the acid resistance and/or lactate production of a yeast cell. The method comprises increasing the expression of a radiation sensitivity complementing kinase in the yeast cell. The expression of a radiation sensitivity complementing kinase can be increased by any suitable method, as described herein, such as by increasing the copy number of a polynucleotide encoding the radiation sensitivity complementing kinase, or by modifying an expression regulatory sequence of a gene encoding the radiation sensitivity complementing kinase. For instance, the expression of a radiation sensitivity complementing kinase can be increased by introducing into the yeast cell an exogenous polynucleotide that encodes the radiation sensitivity complementing kinase; by providing a heterologous promoter operatively linked to a polynucleotide encoding a radiation sensitivity complementing kinase, or both. All other aspects of the method are as described with respect to the genetically engineered yeast cell.
Another aspect of exemplary embodiments provides a composition for producing lactate, wherein the composition includes the yeast cell. The yeast cell is defined the same as described above. The composition may further comprise components useful for producing lactate, such as a cell culture medium, carbon source (e.g., glucose), and other components used for cell culture.
Another aspect of exemplary embodiments provides a method of producing lactate, wherein the method includes culturing the yeast cell. The yeast cell is defined the same as described above.
The culturing of the yeast cell may be performed in a suitable medium under suitable culturing conditions known in the art. One of ordinary skill in the art may suitably change a culture medium and culturing conditions according to the microorganism selected. A culturing method may be batch culturing, continuous culturing, or fed-batch culturing. The yeast cell is the same as described above.
The culture medium may include various carbon sources, nitrogen sources, and trace elements.
The carbon source may be, for example, one or more carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, or cellulose; fat such as soybean oil, sunflower oil, castor oil, or coconut oil; fatty acid such as palmitic acid, stearic acid, or linoleic acid; alcohol such as glycerol or ethanol; organic acid such as acetic acid, and/or a combination thereof. The culturing may be performed by having glucose as the carbon source. The nitrogen source may be an organic nitrogen source such as peptone, yeast extract, beef stock, malt extract, corn steep liquor (CSL), or soybean flour, or an inorganic nitrogen source such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, or a combination thereof. The culture medium is a supply source of phosphorus and may include, for example, potassium dihydrogen phosphate, dipotassium phosphate, and corresponding sodium-containing salt thereof, and a metal salt such as magnesium sulfate or iron sulfate. Also, amino acid, vitamin, a suitable precursor, or the like may be included in the culture medium. The culture medium or individual component may be added to a culture medium solution in a batch or continuous manner.
Also, pH of the culture medium solution may be adjusted by adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium solution by using a suitable method during the culturing process. Also, an antifoaming agent such as fatty acid polyglycol ester may be used during the culturing process to inhibit the generation of bubbles.
The yeast cell may be cultured under an aerobic, microaerobic, or anaerobic condition. In some embodiments, the microaerobic condition refers to a culturing condition in which oxygen is dissolved in the medium at a lower level than that in the atmosphere. For example, the lower oxygen concentration level may be about 0.1% to about 10%, about 1% to about 9%, about 2% to about 8%, about 3% to about 7%, or about 4% to about 6% of a saturated dissolved oxygen in the atmosphere. Also, the microaerobic condition may include maintaining a dissolved oxygen (DO) concentration of the medium in a range of about 0.9 ppm to about 3.6 ppm, for example, about 0.9 ppm to about 3.6 ppm. A temperature for the culturing may be in a range of, for example, about 20° C. to about 45° C. or about 25° C. to about 45° C. A period of time for the culturing may be continued until a desired amount of lactate is obtained. The method of producing lactate may include collecting or isolating lactate from the culture.
The collecting of lactate from the culture may be performed by using a separation and purification method known in the art. The collecting of lactate may be performed by centrifugation, ion-exchange chromatography, filtration, precipitation, extraction, distillation, or combination thereof. For example, the culture may be centrifuged to separate biomass, and a supernatant thus obtained may be separated by ion-exchange chromatography.
According to an aspect of exemplary embodiments, the yeast cell may be an acid-resistant cell, and in addition, may produce lactate at high concentration and a high yield.
According to an aspect of exemplary embodiments, the method of producing lactate may be used to product lactate at a high concentration and a high yield.
Hereinafter, the present invention will be described in further detail with reference to the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1. Manufacture of Yeast Cell with Improved Capability of Producing Lactate
(1) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh)
(1.1) Manufacture of Vector for Pdc1 Deletion and Ldh Introduction
In order to block a pathway from pyruvate to ethanol through acetaldehyde in S. cerevisiae CEN.PK2-1D, a gene that encodes a pyruvate decarboxylase1 (pdc1) was deleted. At the same time, in order to express an Ldh derived from Pelodiscus sinensis japonicas, a pdc1 gene was deleted by substituting the pdc1 gene with an ‘ldh cassette’. As used herein, unless stated otherwise, the term “cassette” refers to a unit sequence from which a protein may be expressed, where the cassette includes promoters, coding sequences, and terminators that are operably linked with each other.
In detail, to manufacture a vector including an ‘ldh cassette’, PCR was performed by using a genomic DNA of S. cerevisiae as a template and a primer set of SEQ ID NOS: 45 and 46 as primers to obtain a CCW12 promoter sequence (SEQ ID NO: 42) and an ‘ldh gene (SEQ ID NO: 10)’. The CCW12 promoter sequence (SEQ ID NO: 42) and the ldh gene (SEQ ID NO: 10) were each digested with SacI/XbaI and BamHI/SalI, and linked to a pRS416 vector (ATCC87521), which was digested with the same enzymes, i.e., SacI/XbaI and BamHI/SalI. The pRS416 vector was a yeast centromere shuttle plasmid with a T7 promoter, ampicillin resistance in bacteria, a URA3 cassette in yeast (a selectable marker), and a restriction enzyme cloning.
Next, PCR was performed on the vector thus obtained using a pCEP4 plasmid (invitrogen, Cat. no. V044-50) as a template and a primer set of SEQ ID NOS: 33 and 34 as primers to obtain amplification product, i.e., was a ‘hygromycin B phosphotransferase (HPH) cassette’ sequence (SEQ ID NO: 49). The HPH cassette sequence was digested with SacI, and then, linked to the vector designed with the same enzyme to prepare a vector p416-ldh-HPH including the ‘ldh cassette’. The pCEP4 plasmid used herein is an episomal mammalian expression vector that uses a cytomegalovirus (CMV) immediate early enhance/promoter for high level transcription of recombinant genes inserted into the multiple cloning site. The pCEP4 plasmid also carries the hygromycin B resistance gene for stable selection in transfected cells. Here, an ‘ldh cassette’ includes an ldh gene and its regulatory region, and thus the ldh cassette refers to a region that allows the ldh gene to be expressed. The ldh gene was transcripted under the control of the CCW 12 promoter. Also, the ‘HPH cassette’ includes a hygromycin B resistance gene and its regulatory region, and thus the HPH cassette refers to a region that allows the hygromycin B resistant gene to be expressed.
In order to prepare a pdc1 deletion cassette, PCR was performed using p416-ldh-HPH as a template and a primer set of SEQ ID NOS: 50 and 51 as primers to prepare a ldh gene fragment and a pUC57-Ura3HA vector (DNA2.0 Inc.; SEQ ID NO: 52). The ldh gene fragment and pUC57-Ura3HA vector were each digested with SacI and then linked to each other to prepare a pUC-uraHA-ldh vector. PCR was performed using sequences of SEQ ID NOS: 53 and 54 having the homologous sequence with the pdc1 gene as primers to amplify the pdc1 deletion cassette from the pUC-uraHA-ldh vector. 1 to 41 of SEQ ID NO: 53 and 1 to 44 of SEQ ID NO: 54 denote sites to be homologously recombined with the homologous chromosomes of S. cerevisiae and substituted with the pdc1 gene.
(1.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh)
The pdc1 deletion cassette prepared in (1.1) was introduced to S. cerevisiae (CEN.PK2-1D, EUROSCARF accession number: 30000B). The introduction of the pdc1 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the pdc1 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 55 and 56 as primers on the cells thus obtained to confirm deletion of pdc1. Therefore, deletion of the pdc1 gene and introduction of the ldh gene were confirmed. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh) was manufactured.
(2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh)
(2.1) Manufacture of Vector for Cyb2 Deletion
In order to block a pathway from lactate to pyruvate, a cyb2 gene was deleted from the S. cerevisiae CEN.PK2-1D (Δpdc1::ldh) prepared in (1).
In detail, PCR was performed using the pUC-uraHA-ldh vector prepared in (1.1) as a template and cyb2 homologous recombinant sequences of SEQ ID NOS: 57 and 58 as primers to obtain a cyb2 deletion cassette. 1 to 45 of SEQ ID NO: 57 and 1 to 45 of SEQ ID NO: 58 denote sites to be homologously recombined with chromosomes of S. cerevisiae and substituted with the cyb2 gene.
(2.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh)
The cyb2 deletion cassette prepared in (2.1) was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh). The introduction of the cyb2 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the cyb2 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 59 and 60 as primers on the strain thus obtained to confirm deletion of cyb2. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh) was manufactured.
(3) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh)
(3.1) Manufacture of Vector for Gpd1 Deletion
In order to block a pathway from DHAP to glycerol-3-phosphate, a gene encoding a glycerol-3-phosphate dehydrogenase 1 (gpd1) was deleted from the S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh) prepared in (2).
In particular, PCR was performed using the pUC-uraHA-ldh vector prepared in (1.1) as a template and gpd1 homologous recombinant sequences of SEQ ID NOS: 61 and 62 as primers to obtain a gpd1 deletion cassette. 1 to 50 of SEQ ID NO: 61 and 1 to 50 of SEQ ID NO: 62 denote sites to be homologously recombined with chromosomes of S. cerevisiae and substituted with the gpd1 gene.
(3.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh)
The gpd1 deletion cassette prepared in (3.1) was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh) prepared in (2). The introduction of the pdc1 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the gdp1 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 63 and 64 as primers on the strain thus obtained to confirm deletion of gpd1. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) was manufactured.
S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) has been deposited to Korean Collection for Type Cultures (KCTC) according to the Budapest Treaty on May 30, 2013, and received an accession number KCTC 12415BP.
(4) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2)
(4.1) Manufacture of Vector for Nde1 Deletion
In order to prepare an nde1 deletion cassette, PCR was performed using the pUC-uraHA-ldh vector prepared in (1.1) as a template and a primer set of SEQ ID NOS: 65 and 66 as primers to prepare an nde1 deletion cassette.
(4.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1)
The nde1 deletion cassette prepared in (4.1) was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) prepared in (3). The introduction of the nde1 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the nde1 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 67 and 68 as primers on the cells thus obtained to confirm deletion of nde1. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1) was manufactured.
(4.3) Manufacture of Vector for Nde2 Deletion
In order to prepare an nde2 deletion cassette, PCR was performed using the pUC-uraHA-ldh vector prepared in (1.1) as a template and a primer set of SEQ ID NOS: 69 and 70 as primers to prepare an nde2 deletion cassette.
(4.4) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2)
The nde2 deletion cassette prepared in (4.3) was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1) prepared in (4.2). The introduction of the nde2 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the nde2 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 71 and 72 as primers on the cells thus obtained to confirm deletion of nde2. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2) was manufactured.
(5) Manufacture of S. cerevisiae CEN.PK2-1D (a Pdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2)
(5.1) Manufacture of Vector for Mpc1 Deletion
In order to delete an mpc1 gene according to homologous recombination, PCR was performed using the pUC57-Ura3HA vector prepared in (1.1) as a template and a primer set of SEQ ID NOS: 73 and 74 (i.e., mpc1_del_F and mpc1_del_R) as primers to prepare an mpc1 deletion cassette.
(5.2) Manufacture of Saccharomyces cerevisiae Having Mpc1 Deletion
The mpc1 deletion cassette prepared in (5.1) was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2). The introduction of the mpc1 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the mpc1 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 75 and 76 as primers on the cells thus obtained to confirm deletion of mpc1. As a result, S. cerevisiae CEN.PK2-1 D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1) was manufactured.
(5.3) Manufacture of Vector for Mpc2 Deletion
In order to delete an mpc2 gene according to homologous recombination, PCR was performed using the pUC57-Ura3HA vector prepared in (1.1) as a template and a primer set of SEQ ID NOS: 77 and 78 (i.e., mpc2_del_F and mpc2_del_R) as primers to prepare an mpc2 deletion cassette.
(5.4)) Manufacture of Saccharomyces cerevisiae Having Mpc1 and Mpc2 Deletion
The mpc2 deletion cassette prepared in (5.3) was introduced to S. cerevisiae CEN.PK2-1D(Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1). The introduction of the mpc2 deletion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the mpc2 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 79 and 80 as primers on the cells thus obtained to confirm deletion of mpc2. As a result, S. cerevisiae CEN.PK2-1D(Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2) was manufactured.
(6) Manufacture of S. cerevisiae CEN.PK2-1D(ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+)
(6.1) Manufacture of Vector for Overexpression of LDH Derived from Bos Taurus 
In order to overexpress an L-ldh gene derived from Bos Taurus, the CCW12 promoter sequence (SEQ ID NO: 42) prepared in (1.1) was cleaved by SacI and XbaI, followed by being introduced to a p416-GPD vector (ATCC® 87360TM) of which a GPD promoter was also cleaved by SacI and XbaI, thereby manufacturing a p416-CCW12p vector.
Afterwards, PCR was performed using a genome DNA of the L-ldh gene (SEQ ID NO: 10) derived from Bos taurus as a template and a primer set of SEQ ID NOS: 81 and 82 as primers. Then, the obtained PCR fragments and the prepared p416-CCW12p vector were each cleaved by BamHI and SalI and ligated to each other to prepare a p416-CCW12p-LDH vector. FIG. 1 depicts a p416-CCW12p-LDH vector.
PCR was performed again using the p416-CCW12p-LDH vector as a template and a primer set of SEQ ID NOS: 83 and 84 as primers, and the PCR fragments obtained therefrom and a fabricated pUC19-HIS3 (Appl Environ Microbiol. 2002 May; 68(5):2095-100, FIG. 2) vector were each cleaved by SacI and ligated to each other to prepare a pUC19-CCW12p-LDH-HIS3 vector (FIG. 3). PCR was performed again using the pUC19-CCW12p-LDH-HIS3 vector as a template and a primer set of SEQ ID NOS: 85 and 86 as primers, so as to prepare a cassette to be introduced to a TRP1 site. Then, the expression cassette including the L-ldh gene derived from Bos taurus was able to be inserted to the TRP1 gene, and that is, the TRP1 gene was deleted while the L-ldh gene was inserted.
(6.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+)
The expression cassette prepared in (6.1) including the L-ldh gene derived from Bos Taurus was introduced to S. cerevisiae CEN.PK2-1D(Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2) prepared in (5). The introduction of the expression cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a histidine (his)-free minimal solid medium (YSD, 6.7 g/L yeast nitrogen base without amino acids, 1.4 g/L Amino acid dropout mix (-his)) to allow the trp1 ORF on the chromosome to be substituted with the expression cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 87 and 88 as primers on the cells thus obtained to confirm deletion of the trp1 gene and introduction of the L-ldh gene. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+) was manufactured.
(7) Manufacture of S. cerevisiae CEN.PK2-1D(ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1 ΔNde2, ΔMpc1 ΔMpc2, Ldh+, Pcyc1PYC1)
In order to manufacture a strain with decreased pyc1 activity, replacement of the pyc1 promoter (Ppyc1) with a promoter whose expression level is lower than expression level of the Ppyc1 was performed as follows. The promoter whose expression level is lower than expression level of the Ppyc1 may be a CYC1 promoter (Pcyc1), a LEUM promoter (Pleum), or a combination thereof.
(7.1) Preparation of Pcyc1 Segment and Manufacture of a Recombinant Vector
In order to obtain DNA fragments including the CYC1 promoter (Pcyc1) (SEQ ID NO: 89), a chromosomal DNA (gDNA) of CEN.PK2-1D which is a wild strain of Saccharomyces cerevisiae was extracted by using Genomic-tip system manufactured by Qiagen company. Then, PCR was performed by using PCR HL premix kit (BIONEER company, hereinafter, the same kit was used) using the gDNA as a template. Following PCR, amplification of the Pcyc1 was performed using the PCR products that were obtained by using a primer set of SEQ ID NOS: 90 and 91 and then cleaved by EcoRI to prepare DNA fragments (hereinafter, referred to as “Pcyc1 cassette”), wherein the DNA fragments were then subjected to gel electrophoresis at 0.8% agarose gel and elution. Afterwards, a pUC19-Trp1 vector plasmid (Appl Environ Microbiol. 2002 May; 68(5):2095-100) and the obtained Pcyc1 cassette were treated by a restriction enzyme, EcoRI, and ligated to each other to manufacture a pUC19-Trp1-Pcyc1 vector. FIG. 4 depicts the pUC19-Trp1-Pcyc1 vector. The pUC19-TRP1-Pcyc1 was used to replay the pyc1 promoter (Ppyc1) to the cyc1 promoter (Pcyc1) according to homologous recombination. Then, PCR was performed again using the pUC19-TRP1-Pcyc1 as a template and a primer set of SEQ ID NOS: 92 and 93 as primers so as to manufacture a cassette to replace the Ppyc1 with Pcyc1.
(7.2) Manufacture of Strain Replacing Ppyc1 with Pcyc1
The Pcyc1 insertion cassette prepared in (7.1) was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+) prepared in (6). The introduction of the Pcyc1 insertion cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the pyc1 promoter (Ppyc1) ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 94 and 95 as primers on the cells thus obtained to confirm deletion of the pyc1 promoter (Ppyc1) and introduction of the cyc1 promoter (Pcyc1). As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1) was manufactured.
(8) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1)
(8.1) Manufacture of Vector for Overexpression of Jen1
In order to overexpress a Jen1 gene, a coding site of the Jen1 gene was digested with BamHI and XhoI from genome DNA of Saccharomyces cerevisiae CEN.PK2-1D strain, and then, ligated to a p416-CCW12p vector, which was digested with the same enzyme, thereby manufacturing a pRS416-Jen1-CCW12p vector. Here, the Jen1 gene was transcribed by the CCW12 promoter. The Jen1 fragments obtained by PCR using the pRS416-Jen1-CCW12p as a template and the pUC57-Ura3HA vector (SEQ ID NO: 35) were each digested with SacI and ligated to each other to manufacture a pUC-uraHA-Jen1 vector. Then, a cassette for Jen1 insertion was obtained by PCR using the pUC-uraHA-Jen1 vector as a template.
(8.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1)
The cassette prepared in (8.1) for Jen1 insertion was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1) prepared in (7). The introduction of the cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the adh4 ORF on the chromosome to be substituted with the cassette. As a result, S. cerevisiae CEN.PK2-1D(Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2 mpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1) was manufactured.
(9) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔFPS1)
(9.1) Manufacture of Vector for FPS1 Deletion
In order to delete a fps1 gene according to homologous recombination, PCR was performed using a pUC57-ura3HA vector of FIG. 5 as a template and a primer set of SEQ ID NOS: 96 and 97 (i.e., fps1_del_F and fps1_del_R) as primers, so as to manufacture a cassette for fps1 deletion.
(9.2) Manufacture of S. cerevisiae CEN.PK2-1D (a Pdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔFPS1)
The cassette prepared in (9.1) for fps1 deletion was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1). The introduction of the cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in a uracil drop out medium to allow the fps1 ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 98 and 99 as primers on the cells thus obtained to confirm deletion of the fps1gene. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1) was manufactured.
Example 2. Manufacture of S. cerevisiae that Overexpresses an RCK1 Gene
(1) Manufacture of Vector for Overexpression of RCK1
(1.1) Manufacture of Vector for RCK1 Promoter Replacement
In order to overexpress a RCK1 gene, replacement of the RCK1 promoter (PRCK1) of S. cerevisiae CEN.PK2-1D Δpdc1::ldh Δcyb2::ldh Δgpd1::ldh (KCTC 12415BP) with TPI1 promoter (PTPI1) whose expression level is higher than expression level of the PRCK1 was performed as follows. FIG. 5 is a view illustrating a process of manufacturing a strain to which the RCK1 promoter substitution vector is inserted in a parent strain S. cerevisiae. 
In order to obtain DNA fragments including the TPI1 promoter (PTPI1) (SEQ ID NO: 100), a chromosomal DNA (gDNA) of CEN.PK2-1D which is a wild strain of S. cerevisiae was extracted by using Genomic-tip system manufactured by Qiagen company. Then, PCR was performed by using PCR HL premix kit using the gDNA as a template.
Following PCR, amplification of the PTPI1 was performed using a primer set of SEQ ID NOS: 101 and 102 as primers and repeating a cycle 30 times of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 52° C., and elongation for 30 seconds at 72° C. The PCR products were then cleaved by EcoRI to prepare DNA fragments (hereinafter, referred to as “PTPI1 cassette”), wherein the DNA fragments were then subjected to gel electrophoresis at 0.8% agarose gel and elution. Afterwards, a P57 vector plasmid (GenScript)(SEQ ID NO: 103) and the obtained PTPI1 cassette were treated by a restriction enzyme, EcoRI, and ligated to each other to manufacture a p57-PTPI1 vector (SEQ ID NO: 104). FIG. 6 depicts the P57 vector and FIG. 7 depicts the p57-PTPI1 vector.
In order to replace the RCK1 promoter (PRCK1) with the PTPI1 promoter (PTPI1) according to homologous recombination, PCR was performed using the p57-PTPI1 vector as a template and a primer set of SEQ ID NOS: 105 and 106 as primers to prepare a cassette to replace the PRCK1 with the PTPI1.
(1.2) Manufacture of Vector for RCK1 Gene Introduction
In order to prepare a vector including a RCK1 cassette, PCR was performed using a genome DNA of S. cerevisiae as a template and a primer set of SEQ ID NOS: 107 and 108 as primers to obtain a RCK1 ORF sequence and a PGK promoter (SEQ ID NO: 43). Each of the obtained sequence and the promoter was digested with SacI/XbaI and BamHI/SalI, and ligated to a pRS416 vector (ATCC87521) that was digested with the same enzymes, thereby obtaining a p416-PGK-RCK1 vector. FIG. 8 is a view of the p416-PGK-RCK1 vector.
(2) Manufacture of Strain for RCK1 Overexpression
(2.1) Manufacture of S. cerevisiae CEN.PK2-1 D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔPRCK1::PTPI1)
The exchange cassette prepared in Example 2.1.1 was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) prepared in Example 1(3.2). The introduction of the expression cassette was performed by general heat shock transformation. Afterwards, the cells were cultured in uracil drop out medium to allow the RCK1 promoter ORF on the chromosome to be substituted with the cassette.
Then, PCR was performed using a genome of the cells as a template and a primer set of SEQ ID NOS: 109 and 110 as primers on the cells thus obtained to confirm the replacement of the PRCK1 with PTPI1. As a result, S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, ΔPRCK1::PTPI1) was manufactured.
(2.2) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, RCK1+)
The p416-PGK-RCK1 vector prepared in Example 2.1.2 was introduced to S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2 mpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1) prepared in Example 1(8). Such an introduction was performed by heat shock transformation typically used in the art, and afterwards, the cells were cultured in an uracil drop out medium. As a result, it was confirmed that the transformed strain CEN.PK2-1D(Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, RCK1+) was obtained.
(2.3) Manufacture of S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔPRCK1::PTPI1, ΔFPS1, ΔPRCK1::PTPI1)
The exchange-cassette prepared in Example 2.1.1 was introduced to the strain S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1) prepared in Example 1(9). Such an introduction was performed by heat shock transformation typically used in the art, and afterwards, the cells were cultured in an uracil drop out medium and were set to substitute the cassette with RCK1 promoter, ORF, in a chromosome.
In regard to the strain obtained therefrom, PCR was performed by using the cell genome as a template and primers of SEQ ID NO: 109 and 110. Then, the obtained PCR products were subjected to electrophoresis to confirm the switch between PRCK1 and PTPI1. As a result, it was confirmed that the strain S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1, ΔPRCK1:PTPI1) was obtained.
Example 3. Lactate Production by Using Strain with Overexpressed RCK1
(1) Evaluation of Lactate Production by Using Strains S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh) and S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔPRCK1::PTPI1)
S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) (hereinafter, referred to as the strain of Control group 1) prepared in Example 1(3) and S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, ΔPRCK1::PTPI1) prepared in Example 2(2.1) (hereinafter, referred to as the strain of Experiment group 1) were each inoculated in 25 ml of YPD broth including 9% glucose, 1% yeast extracts, and 2% Bacto-peptone, and cultured for 40 hours under anaerobic conditions by stirring the broth at a temperature of 30° C. at a rate of about 90 rpm. In the initial stage of the incubation, the optical density (OD) value of the cells at OD600 nm was 1. Then, the yeast culture was periodically extracted from a flask during the incubation so as to measure OD values and concentrations of lactic acid, glucose, and ethanol. The cell growth in the middle of the incubation was evaluated based on OD values of the cells at OD600 measured by using a spectrophotometer. Here, the concentrations of glucose and lactic acid were analyzed according to high performance liquid chromatography (HPLC).
FIG. 9 is a graphical view that shows concentrations of lactic acid and glucose according to the incubation in Control group 1 and Experiment group 1. Referring to FIG. 9, it was confirmed that the strain of Experiment group 1 in which a RCK1 gene was overexpressed had higher lactic acid producibility than that of the strain of Control group 1.
In addition, in Table 1 below, the concentrations of lactic acid and glucose in the strains of Control group 1 and Experiment group 1 measured after 40 hours of the incubation. It was confirmed that the strain of Experiment group 1 in which a RCK1 gene was overexpressed had higher lactic acid production and yield than that of the strain of Control group 1.












TABLE 1







Glucose





Strain

consumption 
LA
ethanol
Yield


name
OD600
(g/L)
(g/L)
(g/L)
(%)




















Control
10.5
84.52 ± 4.90
34.14 ± 1.95 
23.28 ± 1.58
40.39


group 1







Experiment
9.6
84.15 ± 4.70
36.61 ± 2.04
23.10 ± 1.17
43.50


group 1









(2) Evaluation of Lactate Producibility in Strains S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1) and S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, RCK1+)
S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1) prepared in Example 1(8) (hereinafter, referred to as ‘the strain of Control group 2’) and S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, RCK1+) prepared in Example 2(2.2) (hereinafter, referred to as ‘the strain of Experiment group 2’) were each inoculated in 25 ml of 2×YSD/NB broth including 8% glucose, 13.4 g/L yeast nitrogen base, and yeast synthetic dropout without uracil, and cultured for 40 hours under anaerobic conditions by stirring the broth at a temperature of 30° C. at a rate of about 90 rpm. In the initial stage of the incubation, the OD value of the cells at OD600 nm was 1. Then, the yeast culture was periodically extracted from a flask during the incubation so as to measure OD values and concentrations of lactic acid, glucose, and ethanol. The cell growth in the middle of the incubation was evaluated based on OD values of the cells at OD600 measured by using a spectrophotometer. Here, the concentrations of glucose and lactic acid were analyzed according to HPLC.
FIG. 10 is a graphical view that shows concentrations of lactic acid and glucose according to the incubation in Control group 2 and Experiment group 2. Referring to FIG. 10, it was confirmed that the strain of Experiment group 2 in which a RCK1 gene was overexpressed had higher lactic acid producibility and consumption of glucose than those of the strain of Control group 2.
In addition, in Table 2 below, the concentrations of lactic acid and glucose in the strains of Control group 2 and Experiment group 2 measured after 40 hours of the incubation. It was confirmed that the strain of Experiment group 2 in which a RCK1 gene was overexpressed had greater cell growth and higher glucose consumption and lactic acid production than the strain of Control group 2.












TABLE 2







Glucose







consumption
LA
Ethanol
Yield


Strain name
OD600
(g/L)
(g/L)
(g/L)
(%)







Control group
7.65
65.8 ± 1.5
33.9 ± 0.6 
13.2 ± 0.48
51.52


2







Experiment
8.18
72.5 ± 0.5
  37 ± 0.35
16.1 ± 0.31
50.97


group 2









(3) Evaluation of Lactate Producibility in Strains S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔFPS1) and S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔFPS1, ΔPRCK1::PTPI1)
S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1) prepared in Example 1(9) (hereinafter, referred to as ‘the strain of Control group 3’) and S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1, ΔPRCK1::PTPI1) prepared in Example 2(2.3) (hereinafter, referred to as ‘the strain of Experiment group 3’) were each inoculated in 25 ml of YPD broth including 12% glucose, 1% yeast extracts, and 2% Bacto-peptone, and cultured for 40 hours under anaerobic conditions by stirring the broth at a temperature of 30° C. at a rate of about 90 rpm. In the initial stage of the incubation, the OD value of the cells at OD600 nm was 1. Afterwards, 3% glucose was added to the YPD broth after 24 hours of the incubation. Then, the yeast culture was periodically extracted from a flask during the incubation so as to measure OD values and concentrations of lactic acid, glucose, and ethanol. The cell growth in the middle of the incubation was evaluated based on OD values of the cells at OD600 measured by using a spectrophotometer. The concentrations of glucose and lactic acid were analyzed according to HPLC.
FIG. 11 is a graphical view that shows concentrations of lactic acid and glucose according to the incubation in Control group 3 and Experiment group 3. Referring to FIG. 11, was confirmed that the strain of Experiment group 3 in which a RCK1 gene was overexpressed had higher lactic acid production than the strain of Control group 3.
In addition, in Table 3 below, the concentrations of lactic acid and glucose in the strains of Control group 3 and Experiment group 3 measured after 40 hours of the incubation. It was confirmed that the strain of Experiment group 3 in which a RCK1 gene was overexpressed had higher glucose consumption, lactic acid production, and yield than the strain of Control group 3. Here, the lactic acid production of the strain of Experiment group 3 in which a RCK1 gene was overexpressed was improved about 16.9% as compared to that of the strain of Control group 3 in which a RCK1 gene was not overexpressed.












TABLE 3







Glucose





Strain

consumption
LA
Ethanol
Yield


name
OD600
(g/L)
(g/L)
(g/L)
(%)




















Control
10.0
90.7 ± 1.50
33.6 ± 0.90 
19.3 ± 0.09 
37.05


group 3







Experiment
9.4
93.9 ± 1.50
39.3 ± 1.00
22.4 ± 0.65
41.80


group 3









Example 4. Measurement of Acid-Resistant Properties by Using Stain in which RCK1 Gene is Overexpressed
RCK1 genes were set to be overexpressed in a yeast cell, and it was observed how such overexpression affects the acid-resistant properties of the yeast cell.
(1) Evaluation of Acid-Resistant Properties of Strains S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh) and S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔPRCK1::PTPI1)
The strains prepared in Control group 1 (CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh; KCTC 12415BP) and Experiment group 1 (CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, ΔPRCK1::PTPI1) were each inoculated in a YPD medium including 8% glucose, 1% yeast extracts, and 2% Bacto-peptone, and cultured for 40 hours in total under anaerobic conditions by stirring the medium at a temperature of 30° C. at a rate of about 90 rpm. Here, to 2.5% lactic acid having a pH of 2.95 was added to the YPD medium. In the initial stage of the incubation, the OD value of the cells at OD600 nm was 1. Then, the yeast culture was periodically extracted from a flask during the incubation so as to measure OD values and concentrations of lactic acid and glucose.
FIG. 12 is a graphical view that shows concentrations of lactic acid and glucose according to the incubation in Control group 1 and Experiment group 1. Referring to FIG. 12, it was confirmed that the strain of Experiment group 1 in which a RCK1 gene was overexpressed had higher lactic acid production than that of the strain of Control group 1. As lactic acid having a pH 2.95 was included in the media, the results suggest the strain of Experiment group 1 was more resistant to acid than the control group.
In addition, in Table 4 below, the concentrations of lactic acid and glucose in the strains of Control group 1 and Experiment group 1 measured after 40 hours of the incubation. It was confirmed that the strain of Experiment group 1 in which a RCK1 gene was overexpressed had higher lactic acid production and yield than the strain of Control group 1.













TABLE 4









Glucose







consumption
LA
Yield



Strain name
OD600
(g/L)
(g/L)
(%)









Control group 1
5.2 ± 0.0
41.0 ± 1.0
7.7 ± 0.5
18.68



Experiment
5.1 ± 0.0
40.3 ± 0.2
9.8 ± 0.4
23.34



group 1










(2) Evaluation of Acid-Resistant Strains S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔFPS1) and S. cerevisiae CEN.PK2-1D (ΔPdc1::Ldh, ΔCyb2::Ldh, ΔGpd1::Ldh, ΔNde1, ΔNde2, ΔMpc1, ΔMpc2, Ldh+, Pcyc1PYC1, ΔAdh4::Pccw12JEN1, ΔFPS1, PRCK1::PTPI1)
Control group 3 (S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1) and Experimental group 3 (S. cerevisiae CEN.PK2-1D (Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh, Δnde1, Δnde2, Δmpc1, Δmpc2, ldh+, Pcyc1PYC1, Δadh4::Pccw12JEN1, ΔFPS1, PRCK1::PTPI1) were each spread on a YPD solid medium and cultured at a temperature of 30° C. for at least 48 hours. Then, a colony obtained therefrom was inoculated in a 2 ml YPD broth containing 20 g/L of glucose and cultured for 24 hours in total under aerobic conditions by stirring the broth at a temperature of 30° C. at a rate of about 230 rpm. Cell growth was measured at absorbance of 600 nm, and upon dilution of the cultured cells, 10 uL of sterile water containing 10, 102, 103, and 104 colonies, was inoculated in a medium containing lactic acid so as to confirm the viability and growth of the yeast cells.
FIG. 13 shows the results of the viability of yeast cells cultured in an acidic YPD medium prepared with 45 g/L of lactic acid and having a pH of 3.5. Referring to FIG. 13, Experiment group 3 in which RCK1 was overexpressed exhibited greater viability than Control group 3 under the acidic conditions.
The results show that the yeast strain in which a RCK1 gene was overexpressed was unexpectedly more resistant to acid than a yeast in which RCK1 was not overexpressed.
It should be understood that the exemplary embodiments described therein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.
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.
While one or more embodiments of the present invention have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

## Claims

1. A recombinant acid-resistant yeast cell comprising increased radiation sensitivity complementing kinase (RCK) activity as compared to the RCK activity of a parent cell of the recombinant yeast cell, wherein the yeast cell comprises a genetic modification that increases the RCK activity,
wherein an activity of JEN1 is increased in the recombinant yeast cell as compared with that of a parent cell of the recombinant acid-resistant yeast cell;
wherein the recombinant acid-resistant yeast cell further comprises a polynucleotide encoding a lactate dehydrogenase; and deletion or disruption of:
a gene encoding a polypeptide that converts pyruvate to acetaldehyde;
a gene encoding a polypeptide that converts lactate to pyruvate;
a gene encoding a polypeptide that converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate;
a gene encoding NDE1 or NDE2, or both a gene encoding NDE1 and a gene encoding NDE2;
a gene encoding a mitochondrial pyruvate carrier (MPC);
a gene encoding a polypeptide that converts pyruvate to oxaloacetate;
a gene encoding a polypeptide that converts acetaldehyde to ethanol; and
a gene encoding Fps1.

2. The recombinant yeast cell of claim 1, wherein the radiation sensitivity complementing kinase is classified under EC 2.7.11.1.

3. The recombinant yeast cell of claim 1, wherein the radiation sensitivity complementing kinase is RCK1 or RCK2.

4. The recombinant yeast cell of claim 1, wherein the radiation sensitivity complementing kinase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1 or 2.

5. The recombinant yeast cell of claim 1 comprising an increased expression level of a polynucleotide encoding the radiation sensitivity complementing kinase as compared to a parent cell of the recombinant yeast cell.

6. The recombinant yeast cell of claim 1 comprising an exogenous polynucleotide encoding the radiation sensitivity complementing kinase.

7. The recombinant yeast cell of claim 1 comprising an increased copy number of a gene encoding the radiation sensitivity complementing kinase in comparison to a parent cell of the recombinant yeast cell, or comprising a modification of an expression regulatory sequence of a gene encoding the radiation sensitivity complementing kinase, which causes the increased activity of a radiation sensitivity complementing kinase.

8. The recombinant yeast cell of claim 7 comprising an increased copy number of a gene encoding the radiation sensitivity complementing kinase in comparison to a parent cell of the recombinant yeast cell, wherein the increased copy number is due to the presence of an exogenous gene encoding the radiation sensitivity complementing kinase or amplification of an endogenous gene encoding the radiation sensitivity complementing kinase.

9. The recombinant yeast cell of claim 1, wherein the yeast cell belongs to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Zygosaccharomyces, Shizosaccharomyces, or Saccharomycopsis.

10. The recombinant yeast cell of claim 1 wherein the polypeptide that converts pyruvate to acetaldehyde is classified as EC 4.1.1.1, the polypeptide that converts lactate to pyruvate is classified as EC 1.1.2.3 or EC 1.1.2.4, and the polypeptide that converts DHAP to glycerol-3-phosphate is classified as EC 1.1.1.8.

11. The recombinant yeast cell of claim 1, wherein the polypeptide that converts pyruvate to oxaloacetate is classified as EC 6.4.1.1, and the polypeptide that converts acetaldehyde to ethanol is classified as EC 1.1.1.1.

12. A method of producing lactate, the method comprising:
culturing the yeast cell of claim 1 to produce lactate.

13. The method of claim 12, wherein the method comprises:
collecting lactate from the culture.

14. The method of claim 12, wherein the culturing is performed under a condition within a range of pH 2 to 7.

15. A method for preparing a recombinant acid-resistant yeast cell of claim 1, the method comprising introducing into a yeast cell a genetic modification that increases the expression of a radiation sensitivity complementing kinase (RCK) in the yeast cell,
wherein the yeast cell further comprises or is modified to comprise an exogenous polynucleotide encoding JEN1; a polynucleotide encoding a lactate dehydrogenase; and deletion or disruption of:
a gene encoding a polypeptide that converts pyruvate to acetaldehyde;
a gene encoding a polypeptide that converts lactate to pyruvate;
a gene encoding a polypeptide that converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate;
a gene encoding NDE1 or NDE2, or both a gene encoding NDE1 and a gene encoding NDE2;
a gene encoding a mitochondrial pyruvate carrier (MPC);
a gene encoding a polypeptide that converts pyruvate to oxaloacetate;
a gene encoding a polypeptide that converts acetaldehyde to ethanol; and
a gene encoding Fps1.

16. The method of claim 15, wherein the expression of a radiation sensitivity complementing kinase is increased by increasing the copy number of a polynucleotide encoding the radiation sensitivity complementing kinase, or by modifying an expression regulatory sequence of a gene encoding the radiation sensitivity complementing kinase.

17. The method of claim 15, wherein the expression of a radiation sensitivity complementing kinase is increased by introducing into the yeast cell an exogenous polynucleotide that encodes the radiation sensitivity complementing kinase.