Recombinant yeast for producing 2,3-butanediol including pyruvate decarboxylase derived from candida tropicolis and method for producing 2,3-butanediol using the same

Abstract

Provided is a recombinant Saccharomyces cerevisiae for producing 2,3-butanediol, wherein all GPD1 and GPD2 genes involved in glycerol biosynthesis are removed and a gene encoding NADH oxidase is introduced, and wherein pyruvate decarboxylase activity is inactivated and Candida tropicalis PDC1 gene encoding Candida tropicalis pyruvate decarboxylase 1-is introduced, and wherein expression of the Candida tropicalis PDC1 gene is regulated by a GPD2 promoter.

Claims

1. A recombinant Saccharomyces cerevisiae for producing 2,3-butanediol, wherein GPD1 and GPD2 genes encoding glycerol-3-phosphate dehydrogenase are removed and a gene encoding an NADH oxidase is introduced in the recombinant Saccharomyces cerevisiae, wherein a pyruvate decarboxylase gene is inactivated and a Candida tropicalis PDC1 gene encoding Candida tropicalis pyruvate decarboxylase 1 is introduced in the recombinant Saccharomyces cerevisiae, wherein the Candida tropicalis PDC1 gene is operably linked to a GPD2 promoter, and wherein the recombinant Saccharomyces cerevisiae is transformed so as to express acetolactate synthase, is transformed so as to express acetolactate decarboxylase, and is transformed so as to express butanediol dehydrogenase.

2. The recombinant Saccharomyces cerevisiae according to claim 1, wherein the transformation so as to express the acetolactate synthase is carried out by introducing an alsS gene encoding alpha-acetolactate synthase, the transformation so as to express acetolactate decarboxylase is carried out by introducing an alsD gene encoding alpha-acetolactate decarboxylase, and the transformation so as to express butanediol dehydrogenase is carried out by overexpressing a Saccharomyces cerevisiae BDH1 gene encoding Saccharomyces cerevisiae 2,3-butanediol dehydrogenase.

3. The recombinant Saccharomyces cerevisiae according to claim 1, wherein the inactivation of a pyruvate decarboxylase gene is carried out by partially disrupting or knocking out one or more genes selected from PDC1, PDC5 and PDC6.

4. The recombinant Saccharomyces cerevisiae according to claim 1, wherein the NADH oxidase is a Lactobacillus lactis NADH oxidase.

5. The recombinant Saccharomyces cerevisiae according to claim 1, wherein the gene encoding an NADH oxidase is inserted into a p426GPD plasmid, which is a multi-copy plasmid, and wherein the gene encoding an NADH oxidase is operably linked to a TDH3 promoter.

6. A method for producing 2,3-butanediol comprising culturing the recombinant Saccharomyces cerevisiae of claim 1 in a medium supplemented with glucose to thereby produce 2,3-butanediol.

7. The method according to claim 6, wherein the method is carried out while continuously supplying oxygen.

8. The method according to claim 7, wherein the continuous supply of oxygen is carried out by continuously feeding oxygen such that an amount of oxygen supplied in the middle stage of fermentation is lower than an amount of oxygen supplied in an initial stage of fermentation.

9. The method according to claim 6, wherein the culture is fed-batch culture comprising continuously supplying glucose.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1A is a graph showing a result of fermentation with a strain that produces 2,3-butanediol (control group) while not expressing PDC, FIG. 1B is a graph showing a result of fermentation of the strain expressed by CtPDC1, and FIG. 1C is a graph showing a result of fermentation of the strain expressed by KmPDC1;

(3) FIG. 2 shows a result of measurement of a Pdc titer of a strain expressing pyruvate decarboxylase derived from Candida tropicalis under various expression conditions (type of promoter and the number of copies);

(4) FIG. 3 is a graph showing yields of products (ethanol, glycerol and 2,3-butanediol) by PDC-expressing strains having various expression conditions;

(5) FIG. 4A is a graph showing a result of 2,3-butanediol fermentation by the control group for 120 hours and FIG. 4B is a graph showing a result of 2,3-butanediol fermentation by strain BD5 G1CtPDC1 for 120 hours;

(6) FIG. 5 is a graph showing a result of production of 2,3-butanediol by strain BD5_G1CtPDC1 measured by fed-batch culture;

(7) FIG. 6 shows results of in vitro titration of strains expressing NADH oxidase. Con: BD5_p426TDH3, G1: BD5_p406GPD2_L1nox, C2: BD5_p426CYC1_L1nox, T1: BD5_p406TDH3_L1nox, G2: BD5_p426GPD2_L1nox, T2: BD5p426TDH3_L1nox;

(8) FIG. 7 shows a result of determination of change in fermentation behaviors of 2,3-butanediol by NADH oxidase-expressing strain through batch culture. A: BD5_p426TDH3 (control group), B: BD5p426TDH3L1nox.

(9) FIG. 8 shows a change in 2,3-butanediol fermentation behaviors by strain BD5_Ctnox depending on the amount of oxygen supplied. A) 25% air injection, B) 50% air injection, C) 100% air injection;

(10) FIG. 9 shows a result of measurement of changes in concentrations of NADH and NAD.sup.+, intracellular coenzymes, depending on oxygen supply amount;

(11) FIG. 10 is a fed-batch culture profile using strain BD5_Ctnox;

(12) FIG. 11 is a map of a plasmid for Cas9 expression;

(13) FIG. 12 is a schematic diagram showing a point mutation process for removing GPD1 genes;

(14) FIG. 13 is a schematic diagram showing a point mutation process for removing GPD2 genes;

(15) FIG. 14 shows a sequence of GPD1 genes, activity of which is removed, wherein the mutated site is underlined;

(16) FIG. 15 shows a sequence of GPD2 genes, activity of which is removed, wherein the mutated site is underlined;

(17) FIG. 16 shows a fermentation profile of GPD gene-removed strains. A: BD5p426TDH3_L1nox, B: BD5_T2nox_dGPD1, C: BD5_T2nox_dGPD2; D: BD5_T2nox_dGPD1dGPD2; and

(18) FIG. 17 shows a high-concentration 2,3-butanediol fermentation profile using strain BD5_T2nox_dGPD1dGPD2. A: batch culture, B: fed-batch culture.

(19) FIG. 18 shows the Reference Diagram which was prepared to show the result for the search for PDC genes with low activity.

(20) FIG. 19 shows the Reference Diagram which was prepared to show that by removing both GPD1 and GPD2 genes, 2,3-butanediol can be produced with high purity, high yield and high productivity, while completely inhibiting production of glycerol.

DETAILED DESCRIPTION OF THE INVENTION

(21) The present invention focuses on development of a recombinant yeast for producing 2,3-butanediol at high productivity using a metabolic engineering method and production of 2,3-butanediol at high productivity from glucose using the recombinant yeast.

(22) In order to suitably control the activity of Pdc encoding the pyruvate decarboxylase enzyme, PDC genes with low activity were searched for. As a result, a strain for producing 2,3-butanediol with high yield was established by cloning the PDC1 (CtPDC1) gene derived from Candida tropicalis, that is, the nucleic acid sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2, and expressing a single copy of the same (see Reference Diagram, FIG. 18).

(23) Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples. The scope of the present invention is not limited to the following examples and includes modifications of the technical concept equivalent thereto.

Production Example 1: Establishment of 2,3-Butanediol-Producing Strains into which Disruption of PDC1, PDC5 and PDC6 Genes and 2,3-Butanediol Biosynthetic Pathway are Introduced

(24) In case of wild yeast, pyruvate produced as a glucose metabolite is mostly converted into ethanol by pyruvate decarboxylase and alcohol dehydrogenase. Thus, it is necessary to prevent conversion of pyruvate into ethanol in order to produce 2,3-butanediol at high yield from yeast.

(25) Thus, in the present invention, the pyruvate decarboxylase genes, PDC1, PDC5 and PDC6, were first disrupted to establish strains in which the activity of pyruvate decarboxylase is completely removed. In order to introduce the 2,3-butanediol biosynthetic pathway into this strain, acetolactate synthase (alsS) and acetolactate decarboxylase (alsD) derived from Bacillus subtilis were introduced into a plasmid containing the CYC1 terminator and the TDH3 promoter of S. cerevisiae, and 2,3-butanediol dehydrogenase (BDH1) of S. cerevisiae was introduced into a plasmid containing the TDH3 promoter and the CYC1 terminator (refer to Korean Patent Laid-Open No. 10-2015-0068581 filed by the present inventors).

Example 1: Search for Pyruvate Decarboxylase (PDC) Gene Having Low Activity

(26) (1) Introduction

(27) The yeast strain, from which the PDC genes were removed, showed a low productivity of 2,3-butanediol due to low cell growth rate and low substrate consumption rate.

(28) Thus, in order to efficiently produce 2,3-butanediol, the activity of pyruvate decarboxylase was suitably regulated to secure a pyruvate substrate, which is a precursor of 2,3-butanediol, and at the same time, PDC genes with low activity were searched for to supply the C2 compound.

(29) (2) Materials and Methods

(30) A. Genes and Plasmids

(31) After extraction of the genomic DNAs from Candida tropicalis, Kluyveromyces marxianus and Saccharomyces cerevisiae, PDC1 (ScpDC1) derived from Candida tropicalis, PDC1 (KmPDC1) derived from Kluyveromyces marxianus, and PDC1 (ScPDC1), PDC5 (ScPDC5) and PDC6 (ScPDC6) genes derived from Saccharomyces cerevisiae were cloned.

(32) As an expression vector for yeast, the “origin” of “2 micron plasmid” and the p426GPD plasmid containing a TDH3 promoter and a CYC1 terminator derived from S. cerevisiae were used.

(33) The PDC gene fragment obtained by PCR was ligated to restriction sites of XmaI and XhoI of the p426GPD plasmid to establish a recombinant vector.

(34) B. Transformation of Yeast

(35) The established recombinant vector was introduced into the PDC1, PDC5 and PDC6-removed yeast strain of Production Example 1 by transformation.

(36) (3) Result

(37) The “in vitro enzyme activity” of the transformed yeast strain was measured to calculate the kinetic constant value of “K.sub.m” or “V.sub.max” from the activity values of the various concentrations of the substrate. The values are shown in Table 1 below.

(38) TABLE-US-00001 TABLE 1 Strain CtPDC1 KmPDC1 ScPDC1 ScpPDC5 ScPDC6 K.sub.m (mM) 2.7 7.7 4.7 9.9 8.2 V.sub.max (mU/mg 107 383 541 437 415 protein)

(39) Test results showed that the activity of the CtPDC1 strain was significantly low, as compared to other Pdc strains. It was considered that control of Pdc activity necessary for optimizing the production of 2,3-butanediol would be possible with the use of strain CtPDC1 with low activity.

Example 2: Measurement of Cell Growth, Glucose Consumption Rate and 2,3-Butanediol Productivity of Strains Expressing CtPDC1 and KmPDC1

(40) (1) Introduction

(41) Cell growth and glucose consumption rates were measured using the Pdc-expressing strains (CtPDC1, KmPDC1) prepared in Example 1, and the productivities of 2,3-butanediol were compared.

(42) (2) Materials and methods

(43) A. Strains and Plasmids

(44) A strain producing 2,3-butanediol, while not expressing Pdc, a strain expressing CtPDC1 and a strain expressing KmPDC1 were used. CtPDC1 and KmPDC1 were expressed using the TDH3 promoter and the CYC1 terminator of S. cerevisiae.

(45) B. Medium and Culture Conditions

(46) A medium containing 6.7 g/L of “yeast nitrogen base w/o nitrogen base”, 1.4 g/L of “an amino acid mixture” and 80 g/L of glucose was used for fermentation. The initial cell concentration was 0.2 g/L, the fermentation temperature was maintained at 30° C. and the stirring speed was 80 rpm.

(47) (3) Result

(48) The control group with no PDC expression had a glucose consumption rate per hour of 0.10 g/L/h, while the CtPDC1-expressing strain had a glucose consumption rate per hour of 0.94 g/L/h, and the KmPDC1-expressing strain had a glucose consumption rate per hour of 1.09 g/L/h.

(49) The strain not expressing PDC had a maximum dry cell concentration of 0.2 g.sub.DCW/L, while the strain expressing CtPDC1 had a maximum dry cell concentration of 2.7 g.sub.DCW/L and the strain expressing KmPDC1 had a maximum dry cell concentration of 2.8 g.sub.DCW/L.

(50) Meanwhile, the 2,3-butanediol and ethanol yields of the strain not expressing PDC were 0.259 g.sub.2,3-butanediol/g.sub.glucose and 0 g.sub.ethanol/g.sub.glucose, respectively, the 2,3-butanediol and ethanol yields of the strain expressing CtPDC1 was 0.185 g.sub.2,3-butanediol/g.sub.glucose and 0.185 g.sub.ethanol/g.sub.glucose, respectively, and the 2,3-butanediol and ethanol yields of the strain expressing KmPDC1 was 0.134 g.sub.2,3-butanediol/g.sub.glucose and 0.275 g.sub.ethanol/g.sub.glucose, respectively.

(51) Meanwhile, the 2,3-butanediol productivity of the strain not expressing PDC was 0.026 g.sub.2,3-butanediol/L/h, the 2,3-butanediol productivity of the strain expressing CtPDC1 was 0.175 g.sub.2,3-butanediol/L/h, and the 2,3-butanediol productivity of the strain expressing KMPDC1 was 0.147 g.sub.2,3-butanediol/L/h

(52) From the above experiments, it could be confirmed that the expression of PDC in the PDC-deficient 2,3-butanediol-producing strain resulted in significant increases in cell growth and glucose consumption rates, and a great increase in productivity (FIG. 1). FIG. 1A is a graph showing a result of fermentation with a strain producing 2,3-butanediol, while not expressing PDC (control group). FIG. 1B is a graph showing a result of fermentation of the strain expressed by CtPDC1. FIG. 10 is a graph showing a result of fermentation of the strain expressed by KmPDC1.

Example 3: Strain Establishment Having Various Expression Conditions of Pyruvate Decarboxylase Derived from Candida tropicalis and “In Vitro Activity Assay”

(53) (1) Introduction

(54) Since the expression level of PDC directly affects the yield and productivity of 2,3-butanediol, optimal CtPDC1 expression strains were established to select optimal CtPDC1 expression strains.

(55) (2) Materials and Methods

(56) A. Establishment of plasmids having various promoters

(57) The PDC1 gene (CtPDC1) derived from C. tropicalis was amplified and inserted into each expression vector (using a CYC1 promoter, a GPD2 promoter-SEQ ID NO: 3, a TDH3 promoter as a promoter, and a CYC1 terminator as a terminator, respectively), and thereby transformed to establish a recombinant yeast strain. The strain to be used for transformation was a strain obtained when the OD value became 3 after being cultured in YNB medium for 2 days.

(58) B. Transformation

(59) Transformation was carried out using a LiAc method, and strains were selected in YNB leu-his-trp-ura-plate medium after transformation. As a result, a strain (BD5_C1CtPDC1) expressed by the CYC1 promoter and a single copy, a strain (BD5_G1CtPDC1) expressed by the GPD2 promoter and a single copy, a strain (BD5_C2CtPDC1) expressed by the CYC1 promoter and multiple copies, and a strain (BD5_T2CtPDC1) expressed by the TDH3 promoter and multiple copies were established.

(60) (3) Results

(61) In order to determine the PDC expression levels of these strains, “in vitro Pdc activity assay” was performed. As a result, the control group did not show a Pdc titer, while the established strains showed various Pdc expression levels (FIG. 2). FIG. 2 shows a result of Pdc titration of a strain expressing pyruvate decarboxylase derived from Candida tropicalis.

(62) (3) Results

(63) In order to determine the PDC expression levels of these strains, “in vitro Pdc activity assay” was performed. As a result, the control group did not show a Pdc titer, while the established strains showed various Pdc expression levels (FIG. 2). FIG. 2 shows a result of Pdc titration of a strain expressing pyruvate decarboxylase derived from Candida tropicalis.

Example 4: Production of 2,3-Butanediol Using Strain Expressing Pyruvate Decarboxylase Derived from Candida Tropicalis

(64) (1) Introduction

(65) In order to determine the fermentation behaviors of the 2,3-butanediol of the strains established in Example 3, fermentation experiments were performed in a YNB medium containing 90 g/L of glucose in the initial stage. As a control group, a strain transformed with a “p426GPD empty vector with no CtPDC1” was used.

(66) (2) Fermentation Method

(67) The initial strain inoculation concentration was 0.2 g/L, the fermentation temperature was maintained at 30° C., and the stirring speed of 80 rpm was maintained in a glass flask.

(68) (3) Results

(69) For the control group, 2,3-butanediol yield was 0.292 g.sub.2,3-butanediol/g.sub.glucose and no ethanol was produced.

(70) In the BD5_C2CtPDC1 and BD5_T2CtPDC1 strains expressing multiple copies of PDC, a large amount of ethanol was produced, and the yields of 2,3-butanediol were decreased to 0.245 g.sub.2,3-butanediol/g.sub.glucose and 0.150 g.sub.2,3-butanediol/g.sub.glucose respectively (FIG. 3). FIG. 3 is a graph showing yields of products by the control group and PDC-expressing strains. FIG. 3 shows that BD5_C1CtPDC1 and BD5_GlCtPDC1 strains exhibit similar 2,3-butanediol production yields, but, non-preferably, BD5_C1CtPDC1 exhibits a high production yield of glycerol as a by-product. Therefore, it could be considered the BD5_G1CtPDC1 strain was more suitable as a 2,3-butanediol producing strain.

(71) Meanwhile, the PDC-expressing strain exhibited increased cell growth and glucose consumption rates. The glucose consumption rate per hour of the control group was 0.26 g.sub.glucose/L/h, while the glucose consumption rate per hour of the BD5_G1CtPDC1 strain was 0.62 g.sub.glucose/L/h. The BD5_G1CtPDC1 strain exhibited a similar 2,3-butanediol production yield to the control group, but the BD5_GlCtPDC1 strain exhibited an increased 2,3-butanediol productivity of 0.181 g/L/h, while the control group exhibited 2,3-butanediol productivity of 0.076 g/L/h (FIG. 4).

(72) FIG. 4A is a graph showing a result of 2,3-butanediol fermentation by control group for 120 hours and FIG. 4B is a graph showing a result of 2,3-butanediol fermentation by BD5_GlCtPDC1 strain for 120 hours.

Example 5: Production of 2,3-Butanediol by Fed-Batch Culture

(73) (1) Introduction

(74) In order to finally determine whether or not the BD5 G1CtPDC1 strain is suitable as a 2,3-butanediol-producing strain, fed-batch culture involving adding glucose during fermentation was performed.

(75) (2) Materials and Methods

(76) The medium used was YP medium (containing 10 g/L of yeast extract and 20 g/L of peptone), the initial glucose concentration was 270 g/L, and a 800 g/L glucose solution was added at the middle stage of fermentation. The initial cell concentration was 2 g/L, the fermentation temperature was maintained at 30° C., 0.5 vvm of air was injected and the stirring speed was maintained at 200 rpm.

(77) (3) Results

(78) 121.8 g/L of 2,3-butanediol was produced for a culture time of 80 hours. At this time, the yield of 2,3-butanediol was 0.329 g.sub.2,3-butanediol/g.sub.glucose and productivity was as high as 1.61 g/L/h (FIG. 5). FIG. 5 is a graph showing production results of 2,3-butanediol measured by fed-batch culture using the BD5_GlCtPDC1 strain.

(79) From these results, it could be seen that the strain introduced with pyruvate decarboxylase derived from Candida tropicalis according to the present invention was suitable for the production of 2,3-butanediol.

(80) The recombinant Saccharomyces cerevisiae strain for producing 2,3-butanediol is introduced with alpha-acetolactate synthase (alsS) and acetalactate decarboxylase (alsD) derived from Bacillus subtilis, in order to introduce a 2,3-butanediol biosynthesis pathway, and over-expresses the 2,3-butanediol dehydrogenase (BDH1) genes possessed by the yeast. In this regard, the present inventors developed, in addition to the basic 2,3-butanediol-producing strain, the strain (“BD5 strain”) wherein PDC1, PDC5 and PDC6, pyruvate decarboxylase genes possessed by the Saccharomyces cerevisiae strain, are disrupted, and furthermore, developed the strain (“BD5_G1CtPDC1”) into which the PDC1 gene encoding pyruvate decarboxylase derived from Candida tropicalis, which is less active than Pdc possessed thereby, is introduced (see Reference Diagram, FIG. 18).

(81) However, in the present invention, the NADH oxidase (NoxE) gene derived from Lactobacillus lactis was further expressed in the yeast strain for producing 2,3-butanediol, and glycerol-3-phosphate dehydrogenase (GPD1, GPD2) genes involved in glycerol biosynthesis were disrupted, to produce a yeast strain for producing 2,3-butanediol (Reference Diagram, FIG. 19).

(82) When the GPD1 or GPD2 gene involved in glycerol biosynthesis is removed using NADH as a coenzyme, glycerol production may be reduced, but NADH is not consumed and accumulates in the cytoplasm. As a result, a problem occurs that the strain does not grow efficiently. However, according to the present invention, NADH can be consumed because NADH oxidase capable of oxidizing NADH is overexpressed.

(83) Therefore, in the present invention, by removing both GPD1 and GPD2 genes, 2,3-butanediol can be produced with high purity, high yield and high productivity, while completely inhibiting production of glycerol (See Reference Diagram, FIG. 19).

(84) Meanwhile, in the present invention, it was confirmed that the amount of oxygen supplied into the incubator can regulate the activity of the strain introduced with NADH oxidase established according to the present invention, and also affected the production of 2,3-butanediol. In addition, 2,3-butanediol production technology with high concentration, high yield and high productivity was developed through optimization of the culture process such as control of sugar concentration and oxygen supply.

(85) Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples and test examples. The scope of the present invention is not limited to the following examples and test examples and includes modifications of the technical concept equivalent thereto.

Example 6: Production of Strain Expressing NADH Oxidase

(86) A yeast strain for producing 2,3-butanediol was prepared through the previous study of the present inventors (Korean Patent Laid-open No. 10-2015-0068581, Korean Patent Application No. 10-2015-0124845) and this strain was used as a parent strain (strain “BD5”) by the present inventors. The parent strain was obtained by removing the pyruvate decarboxylase (Pdc) genes, that is, PDC1, PDC5 and PDC6 from S. cerevisiae strain, and by introducing and reinforcing alpha-acetolactate synthase (AlsS) and alpha-acetolactate decarboxylase (AlsD) derived from Bacillus subtilis, and 2,3-butanediol dehydrogenase (Bdh1) derived from Saccharomyces cerevisiae so as to form a 2,3-butanediol biosynthetic pathway.

(87) Meanwhile, production of plasmids for expression and gene cloning were performed in order to further introduce NADH oxidase into the parent strain. Five types of expression plasmids were produced for expression.

(88) The CYC1 gene promoter, 289 bp, and the GPD2 gene promoter, 1144 bp, of Saccharomyces cerevisiae were inserted into the SacI and BamHI sites of the pRS406 (Mumberg et al., Yeast vectors for heterologous proteins in different genetic backgrounds. Gene 156 (1), 119-122) plasmid, which is a single copy plasmid, and the 655 bp TDD3 gene promoter of Saccharomyces cerevisiae was inserted into the SacI and XbaI sites thereof.

(89) The NADH oxidase gene (SEQ ID NO: 4) cloned from the Lactobacillus lactis subsp. cremoris MG1363 strain by PCR was inserted into five types of expression plasmids in total (p406GPD2, p406TDH3, p426CYC1, p426GPD2, p426TDH3).

(90) Five types of 2,3-butanediol-producing strains (BD5_p406GPD2_L1nox, BD5_p406TDH3_L1nox, BD5_p426CYC1_L1nox, BD5_p426GPD2_L1nox, BD5_p426TDH3_L1nox) expressing NADH oxidase were produced through such a process.

(91) In addition, the pyruvate decarboxylase genes used in the prior art to increase the glucose consumption rate and cell growth rate of the Pdc-deficient yeast strain were introduced. For this purpose, NADH oxidase was expressed using, as a single copy plasmid, a TDH3 promoter having a resistant gene against aureobasidin A as an antibiotic, and a CYC1 terminator, and pyruvate decarboxylase (CtPDC1) derived from Candida tropicalis was expressed in the presence of the GPD2 promoter. The gene encoding pyruvate decarboxylase (CtPDC1) derived from Candida tropicalis is set forth in SEQ ID NO: 1.

(92) Through the above process, strain BD5_Ctnox simultaneously expressing NADH oxidase and pyruvate decarboxylase could be established.

(93) TABLE-US-00002 TABLE 2  Primers used for establishment of plasmids expressing NADH oxidase Restriction Primers site Sequence Cloning of S. cerevisiae promoters F_CYC1P SacI cGAGCTCatttggcgagcgttg (SEQ ID NO: 7) R_CYC1P BamHI cgcGGATCCttagtgtgtgtatttgtg tttgc (SEQ ID NO: 8) F_GPD2P SacI cGAGCTCcaaaaacgacatatctatta tagtg (SEQ ID NO: 9) R_GPD2P BamHI cgcGGATCCctttgagtgcagttgtgt tt (SEQ ID NO: 10) Cloning of L. lactis noxE F_nox BamHI cgcGGATCCaaaatgaaaatcgtagtt atcggta (SEQ ID NO: 11) R_XhoI_nox XhoI ccgCTCGAGtttatttggcattcaaag ct (SEQ ID NO: 12) R_SalI_nox SalI acgcGTCGACtttatttggcattcaaa gct (SEQ ID NO: 13)

(94) The CYC1 promoter is set forth in SEQ ID NO: 5, the GPD2 promoter is set forth in SEQ ID NO: 3, and the TDH3 promoter is set forth in SEQ ID NO: 6.

Example 7: In Vitro Enzyme Titration of Strain Expressing NADH Oxidase

(95) The plasmid for expressing NADH oxidase produced in Example 6 was designed to have different activity. Thus, in vitro enzyme titers were measured to compare the expression levels of NADH oxidase in the produced strains.

(96) About 1×10.sup.9 exponential phase cells cultured in YNB medium (6.7 g/L, amino acid mixture 1.4 g/L) supplemented with 80 g/L of glucose and 0.5 g/L of ethanol were used.

(97) After the intracellular enzymes were extracted using Yeast protein extraction reagent (Y-PER, Thermo Scientific, MA), the supernatant was used for the measurement of NADH oxidase. The measurement of NADH oxidase was carried out at 30° C. using a decreased absorbance at 340 nm through reaction in 50 mM potassium phosphate buffer containing 0.4 mM NADH and 0.3 mM EDTA (pH 7.0). Protein concentrations in crude extracts were measured by the Bradford method. 1 unit was expressed as 1 μmol of NADH oxidized per minute.

(98) Test results show, as can be seen from FIG. 6, that the NADH oxidase activity varies depending on the type of promoter and the number of copies used for expression. The control group was transformed with a BD5 strain having the p426TDH3 vector inserted thereinto (Kim et al., expression of Lactococcus lactis NADH oxidase increases 2,3-butanediol production in Pdc-deficient Saccharomyces cerevisiae, Bioresource Technology 191 (2015) 512-519). The control group had an activity of 4.8 mU/mg protein, while the strain BD5_p406GPD2_L1nox had an activity of 11.2 mU/mg protein, and the strain BD5_p426TDH3_L1nox had an activity of 9153 mU/mg protein, which was 900 times higher than that of the strain BD5p406GPD2L1nox.

(99) FIG. 6 shows the results of in vitro titration of strains expressing NADH oxidase. Con: BD5p426TDH3, G1: BD5_p406GPD2_L1nox, C2: BD5_p426CYC1_L1nox, T1: BD5_p406TDH3_L1nox, G2: BD5_p426GPD2_L1nox, T2: BD5_p426TDH3_L1nox.

Example 8: Determination of Change in Fermentation Behaviors of 2,3-Butanediol According to NADH Oxidase Activity

(100) When Pdc-deficient yeast strains for producing 2,3-butanediol produce 2,3-butanediol from sugars, NADH accumulates in the cytoplasm. In this regard, oxidation of NADH through respiration is limited, which results in production of excessive glycerol as a by-product.

(101) Therefore, in the present invention, introduction of an additional pathway for oxidizing NADH was attempted by introduction of NADH oxidase. If such a prediction is successful, it is possible to reduce accumulation of glycerol as a by-product and increase production of 2,3-butanediol.

(102) In order to identify this, batch fermentation experiments were conducted. The medium containing 80 g/L of glucose and 0.5 g/L of ethanol was used as YNB at the initial stage and the fermentation temperature was maintained at 30° C. Stirring was conducted at a rate of 80 rpm in 50 mL of a working volume in a 250 mL flask. The initial cell inoculation concentration corresponded to an optical density (OD) of 1.0 at a wavelength of 600 nm.

(103) The test results are shown in FIG. 7 and Table 3. Both the control and experimental groups perfectly consumed 80 g/L of glucose within about 76 hours. The control group had a glycerol production yield of 0.278 g.sub.Glycerol/g.sub.Glucose, while the strain BD5_p426TDH3_L1nox had a glycerol production yield of 0.209 g.sub.Glycerol/g.sub.Glucose. In addition, the control group had a 2,3-butanediol production yield of 0.332 g.sub.2,3-butanediol/g.sub.Glucose, while the BD5_p426TDH3_L1nox strain had an increased 2,3-butanediol production yield of 0.367 g.sub.2,3-butanediol/g.sub.Glucose. As such, through the expression of NADH oxidase, the production yield of glycerol was decreased and the production yield of 2,3-butanediol was increased. At this time, as the amount of NADH oxidase expression increased, the effect was improved.

(104) FIG. 7 shows a result of determination of change in fermentation behaviors of 2,3-butanediol by NADH oxidase-expressing strain by batch culture. A: BD5_p426TDH3 (control group), B: BD5_p426TDH3_L1nox.

(105) TABLE-US-00003 TABLE 3 Results of batch fermentation using strain expressing NADH oxidase Value (mean ± standard deviation) BD5 BD5_ BD5 BD5 BD5 BD5 _p406GPD2 p426CYC1 _p406TDH3 _p426GPD2 _p426TDH3 Parameter* _p426TDH3 _Llnox _Llnox _Llnox _Llnox _Llnox DCW 2.09 ± 0.10 1.93 ± 0.07 1.74 ± 0.11 1.55 ± 0.09 1.68 ± 0.04 1.69 ± 0.03 (g/L) Y.sub.glycerol 0.278 ± 0.011 0.266 ± 0.010 0.229 ± 0.001 0.231 ± 0.005 0.216 ± 0.009 0.209 ± 0.004 (g/g) Y.sub.2,3-BD 0.332 ± 0.000 0.338 ± 0.001 0.359 ± 0.000 0.359 ± 0.001 0.364 ± 0.003 0.367 ± 0.002 (g/g) Y.sub.acetoin 0.010 ± 0.001 0.009 ± 0.001 0.012 ± 0.001 0.011 ± 0.001 0.010 ± 0.000 0.012 ± 0.001 (g/g) V.sub.glucose 1.10 ± 0.03 1.11 ± 0.02 1.06 ± 0.01 1.13 ± 0.00 1.07 ± 0.02 1.10 ± 0.00 (g/L .Math. h.sup.−1) P.sub.2,3-BD 0.364 ± 0.011 0.374 ± 0.008 0.381 ± 0.003 0.404 ± 0.002 0.391 ± 0.006 0.403 ± 0.002 (g/L .Math. h.sup.−1)

Example 9: Determination of Change in Fermentation Behavior of 2,3-Butanediol by Oxygen Supply Difference

(106) The NADH oxidase used in this study reacts with NADH using oxygen as a substrate to produce water and NAD.sup.+. Thus, it is possible to control the activity of NADH oxidase through the amount of oxygen supplied to the medium during fermentation, in addition to control of the expression level of NADH oxidase enzyme.

(107) In this Example, the activity of NADH oxidase was changed according to the supply of oxygen, and a batch culture experiment was performed under different oxygen conditions in order to identify the effect of the NADH oxidase activity on the fermentation of 2,3-butanediol.

(108) The medium used was a YP medium (yeast extract 10 g/L, peptone 20 g/L) supplemented with 90 g/L of glucose. The oxygen supply conditions were varied such that 25%, 50% and 100% air was introduced by mixing nitrogen with the air supplied into the medium. The fermentation temperature was maintained at 30° C., and air and a gas mixture of air/nitrogen were fed at 2 vvm and stirred at 500 rpm. A 1 L fermenter was used and the working volume was 500 mL. The strain BD5_Ctnox was used.

(109) Test results are shown in Table 4 and FIG. 8. As shown in Table 8, the production behavior of 2,3-butanediol was changed according to the amount of oxygen supplied. As oxygen supply increased, glycerol production decreased and acetoin production increased. The production yield of 2,3-butanediol was the highest at 50% air. There was no significant difference in cell growth between the three conditions. However, the glucose consumption rate decreased under the condition of 100% air injection, as compared to under the other two conditions.

(110) FIG. 8 shows a change in 2,3-butanediol fermentation behaviors of the BD5_Ctnox strain depending on the amount of oxygen supplied. A) 25% air injection, B) 50% air injection, C) 100% air injection.

(111) TABLE-US-00004 TABLE 4 Changes in 2,3-butanediol fermentation behaviors of BD5_Ctnox strain depending on oxygen supply Glycerol 2,3-BD DCW.sub.max Glycerol Acetoin 2,3-BD yield 2,3-BD Yield productivity Parameters (g/L) (g/L) (g/L) (g/L) (g/g) (g/g) (g/L .Math. h.sup.−1)  25% 3.0 19.2 2.0 31.4 0.216 0.363 1.42  50% 3.3 11.3 2.2 33.1 0.128 0.374 1.44 100% 3.4 3.7 14.2 17.7 0.050 0.237 0.71

Example 10: Measurement of Concentration Change in Intracellular NADH/NAD.SUP.+ Coenzymes Depending on Oxygen Supply Amount

(112) The concentrations of NADH and NAD.sup.+ in the cells were measured using the 20-hour-old cells obtained in Example 9. About 4×10.sup.7 cells were used for measurement and were measured using a kit for NAD.sup.+/NADH measurement (BioAssay Systems, CA).

(113) Test results are shown in FIG. 9. As shown in FIG. 9, when the air ratio was 100%, the concentration of NADH decreased and the concentration of NAD+ increased. That is, as oxygen supply increased, the activity of intracellular NADH oxidase increased, which means that oxygen acts on NADH in the cells to directly oxidize NADH.

(114) FIG. 9 shows a result of measurement of changes in concentrations of NADH and NAD.sup.+, intracellular coenzymes, depending on oxygen supply amount.

Example 11: Fermentation of High-Concentration and High-Productivity 2,3-Butanediol by Controlling Oxygen Supply During Fed-Batch Culture

(115) In order to determine the availability of BD5_Ctnox strain as a strain for mass production of 2,3-butanediol, fed-batch culture was carried out by adding glucose during fermentation. The activity of NADH oxidase was regulated to reduce production of glycerol and the amount of oxygen fed during fermentation was converted in order to maximize production of 2,3-butanediol. YP medium was used as a medium, the initial glucose concentration was 330 g/L, and a 800 g/L glucose solution was added in the middle stage of fermentation. The initial cell inoculation concentration was 2.0 g/L and the fermentation temperature was maintained at 30° C. Oxygen was supplied under the conditions of 2 vvm and 500 rpm from the beginning to the middle of fermentation, and then oxygen was supplied at 1 vvm and 200 rpm. As a result, after the culture time of 78 hours, 154.3 g/L of 2,3-butanediol was produced and the productivity was 1.98 g.sub.2,3-Butanediol/L/h (see FIG. 10 and Table 5). At this time, the 2,3-butanediol production yield was 0.404 g.sub.2,3-Butanediol/g.sub.Glucose.

(116) FIG. 10 is a fed-batch culture profile using strain BD5_Ctnox.

(117) TABLE-US-00005 TABLE 5 Results of fedbatch culture profile using strain BD5_Ctnox. Glucose Glycerol 2,3-BD 2,3-BD DCW.sub.max consumed Glycerol 2,3-BD Ethanol yield yield productivity Parameters (g/L) (g/L) (g/L) (g/L) (g/L) (g/g) (g/g) (g/L .Math. h.sup.−1) BD5_Ctnox 6.2 404.3 32.5 154.3 0.1 0.088 0.404 1.98

Example 12: Preparation of Strain for Producing 2,3-Butanediol from which GPD1 and GPD2 are Removed

(118) Glycerol-3-phosphate dehydrogenase (Gpd) in Saccharomyces cerevisiae is a key metabolic enzyme for glycerol production, which is expressed by the GPD1 and GPD2 genes.

(119) In the present invention, a recombinant yeast strain for producing 2,3-butanediol, from which the GPD1 and GPD2 genes were completely removed, was established in order to completely inhibit production of glycerol during 2,3-butanediol production. For this purpose, the Cas9-CRISPR method was applied and GPD1 and GPD2 genes was inactivated by converting the codons in the middle of ORF of the GPD1 and GPD2 genes, to termination codons. In order to apply the Cas9-CRISPR method, a Cas9 gene sequence was inserted into a plasmid containing the AUR1-C gene resistant to Aureobasidin A, as an antibiotic, and was expressed in the yeast. Guide DNA and repair DNA targeting the mutated parts of GPD1 and GPD2, respectively, were transformed into the 2,3-butanediol-producing NADH oxidase-expressing strain, to produce strains BD5_T2nox_dGPD1, BD5_T2nox_dGPD2, and BD5_T2_nox_dGPD1dGPD2, in which GPD1 and GPD2 were inactivated.

(120) In addition, pyruvate kinase derived from Candida tropicalis was introduced into strain BD5_T2nox_dGPD1dGPD2 expressed by the GPD2 promoter to produce the BD5_T2nox_dGPD1dGPD2_CtPDC1 strain.

(121) FIG. 11 is a map of a plasmid for Cas9 expression. FIG. 12 is a schematic diagram showing a point mutation process for removing GPD1 genes. FIG. 13 is a schematic diagram showing a point mutation process for removing GPD2 genes. FIG. 14 shows a sequence of GPD1 genes, activity of which is removed, wherein the mutated site is underlined. FIG. 15 shows a sequence of GPD2 genes, activity of which is removed, wherein the mutated site is underlined.

Example 13: Production of GPD1 and GPD2-Free Strains with No NADH Oxidase Activity

(122) Strains for producing 2,3-butanediol having no NADH oxidase activity were produced by removing the plasmid for expressing NADH oxidase from BD5_T2nox_dGPD1, BD5_T2nox_dGPD2 and BD5_T2nox_dGPD1dGPD2 strains prepared in Example 12.

(123) For this purpose, BD5_T2nox_dGPD1, BD5_T2nox_dGPD2 and BD5_T2nox_dGPD1dGPD2 strains were repeatedly sub-cultured in a medium containing uracil, which is a marker of plasmid for expressing NADH oxidase, and strains, from which the NADH oxidase plasmid was removed, were selected in a medium containing uracil and in a medium containing no uracil by replica plating.

(124) As a result, in case of the BD5_T2nox_dGPD1 strains, NADH oxidase was removed from five strains out of 16 strains in total, and in case of the BD5_T2nox_dGPD2 strains, NADH oxidase was removed from six strains out of 16 strains (Table 6 and FIG. 16).

(125) However, in the case of the strain BD5_T2nox_dGPD1dGPD2, strains, from which NADH oxidase was removed, could be not obtained from 120 strains in total. This means that NADH oxidase is an essential enzyme for growth of Pdc-deficient Gpd-deficient 2,3-butanediol yeast strains. The final conclusion is that NADH oxidase functions to oxidize additional NADH, which is produced by the removal of GPD gene, into NAD.sup.+, so that the recombinant strain can grow. FIG. 16 shows results of plasmid curing tests of strains BD5_T2nox_dGPD1, BD5_T2nox_dGPD2 and BD5_T2nox_dGPD1_dGPD2.

(126) TABLE-US-00006 TABLE 6 Results of plasmid curing tests of strains BD5_T2nox_dGPD1, BD5_T2nox_dGPD2 and BD5_T2nox_dGPD1_dGPD2. Strains Ura-cell/Total cells BD5_T2nox_dGPD1 5/16 — BD5_T2nox_dGPD2 6/16 — BD5_T2nox_dGPD1_dGPD2 0/24 0/96

Example 14: Fermentation of 2,3-Butanediol Using GPD-Free Strain

(127) Fed-batch culture was carried out using the yeast strain for producing 2,3-butanediol prepared in Example 12. YP medium was used. At the initial stage, 100 g/L of glucose and 0.7 g/L of ethanol were supplied as a carbon source. Cell inoculation concentration, culture temperature, culture conditions and the like were the same as in Example 3.

(128) As a result of the culture, as compared to the glycerol yield (0.166 g.sub.Glycerol/g.sub.Glucose) of control group, the strain, from which GPD1 was removed, had a decreased glycerol yield of 0.086 g.sub.Glycerol/g.sub.Glucose and the strain, from which GPD2 was removed, was decreased to 0.083 g.sub.Glycerol/g.sub.Glucose. In addition, glycerol was not observed in the strain from which both GPD1 and GPD2 were removed, and the yield of 2,3-butanediol was 0.363 g.sub.2,3-butanediol/g.sub.Glucose, which was increased by 10% as compared with the control group (FIG. 16, Table 7).

(129) FIG. 16 shows fermentation profiles of the GPD gene-free strains. A: BD5_p426TDH3_L1nox, B: BD5_T2nox_dGPD1, C: BD5_T2nox_dGPD2, D: BD5_T2nox_dGPD1dGPD2.

(130) TABLE-US-00007 TABLE 7 Fermentation results of GPD gene-free strains Glycerol 2,3-BD 2,3-BD DCW.sub.max Glycerol Acetoin 2,3-BD yield yield productivity Parameters (g/L) (g/L) (g/L) (g/L) (g/g) (g/g) (g/L .Math. h.sup.−1) BD5_T2nox 1.3 15.7 1.9 31.5 0.166 0.333 0.33 BD5_T2nox_dGPD1 1.1 6.4 5.6 22.3 0.086 0.302 0.19 BD5_T2nox_dGPD2 1.1 6.4 5.1 24.3 0.083 0.317 0.20 BD5_T2nox_dGPD1_dGPD2 1.0 0 1.7 16.5 0 0.363 0.14

Example 15: Production of High-Concentration and High-Productivity 2,3-Butanediol by Batch and Fed-Batch Culture and Oxygen Supply Control

(131) In order to identify the industrial applicability of the strain for producing 2,3-butanediol prepared in Example 14, high-concentration 2,3-butanediol was produced by batch and fed-batch culture methods. The strain used was BD5_T2nox_dGPD1dGPD2_CtPDC1 and the initial cell inoculation concentration was 3.4 gDCW/L. The fermentation temperature was maintained at 30° C. For batch culture, 300 g/L of glucose was added to YP medium at the initial stage. For fed-batch culture, 100 g/L of glucose was added and 150 g/L of glucose was added at the middle of fermentation. The initial oxygen supply was 2 vvm, 400 rpm, and the oxygen supply was reduced to 1 vvm, 200 rpm and 2 vvm 300 rpm in the middle of fermentation.

(132) The test results are shown in FIG. 17 and Table 8.

(133) As a result of the batch culture, 237.9 g/L of glucose was consumed from 300 g/L of glucose, resulting in production of 99.4 g/L of 2,3-butanediol therefrom. At this time, the yield of 2,3-butanediol was 0.418 g.sub.2,3-butanediol/g.sub.Glucose, and the productivity thereof was 0.62 g.sub.2,3-Butanediol/L/h.

(134) As a result of the fed-batch culture, 108.6 g/L of 2,3-butanediol was produced during culture for 70 hours. At this time, the yield of 2,3-butanediol was 0.495, which corresponded to 99% of the theoretical yield. By-products of glycerol, acetic acid and ethanol were not produced at all, and acetoin was produced at 3.5 g/L. The productivity of 2,3-butanediol could be improved by minimizing the inhibitory effect of Gpd-removed strains by high-concentration glucose through fed-batch culture. In addition, the amount of oxygen fed into the medium could regulate NADH oxidase activity, thereby contributing to the growth of strains and minimizing production of acetoin by-products.

(135) FIG. 17 shows a high-concentration 2,3-butanediol fermentation profile using BD5_T2nox_dGPD1dGPD2 strain. A: batch culture, B: fed-batch culture.

(136) TABLE-US-00008 TABLE 8 High-concentration 2,3-butanediol fermentation profile using BD5_T2nox_dGPD1dGPD2 strain Glucose Glycerol 2,3-BD 2,3-BD consumed Glycerol Acetoin 2,3-BD Acetate Ethanol yield yield productivity Condition (g/L) (g/L) (g/L) (g/L) (g/L) (g/L) (g/g) (g/g) (g/L .Math. h.sup.−1) Batch 237.9 0.5 11.3 99.4 1.3 0.2 0.002 0.418 0.62 Dumping 235.1 0.2 3.5 108.6 0.1 0 0.001 0.495 1.55

(137) As apparent from the foregoing, methods for producing 2,3-butanediol using conventional yeasts had a limitation in obtaining 2,3-butanediol with low productivity.

(138) However, the yeast strain for producing 2,3-butanediol according to the present invention can synthesize acetyl-CoA, while avoiding production of ethanol by introducing Candida tropicalis-derived Pdc, which is less active than its own pyruvate decarboxylase (Pdc), into the strain, thereby increasing the strain growth rate and the substrate consumption rate and ultimately greatly improving productivity of 2,3-butanediol.

(139) That is, conventional Pdc-deficient strains did not produce acetyl-CoA in addition to ethanol biosynthesis due to the deficiency of Pdc, such that the strains could not grow efficiently. The present invention can solve this problem by introducing Candida tropicallis-derived Pdc into cells of the strain. Thus, according to the present invention, a high concentration of 2,3-butanediol can be produced with high productivity.

(140) Conventional methods for producing 2,3-butanediol using recombinant Saccharomyces cerevisiae (yeast) result in production of a large amount of glycerol as a by-product, in addition to the production of 2,3-butanediol. However, when the recombinant Saccharomyces cerevisiae strain according to the present invention is used, 2,3-butanediol can be produced with high purity, high yield and high productivity, while inhibiting production of glycerol.

(141) Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.