KR20170028055A - Recombinant Saccharomyces cerevisiae for the production of 2,3-butanediol with pyruvate decarboxylase from Candida tropicalis and method for the production of 2,3-butanediol therefrom - Google Patents

Abstract

The present invention relates to a recombinant yeast for producing 2,3-butanediol, and to a method for producing 2,3-butanediol using the same. By introducing pyruvate decarboxylase derived from candida tropicalis which has lower activity than originally retaining pyruvate decarboxylase, ethanol, which acts as byproducts when producing 2,3-butanediol, is not produced and the synthesis of acetyl-CoA is possible which enables an increase in a growth rate of the bacteria and a rate of substrate consumption. Ultimately, the productivity of 2,3-butanediol can be greatly improved.

Description

Recombinant yeast for producing 2,3-butanediol having pyruvate decarboxylase derived from Candida tropicallis and method for producing 2,3-butanediol using the same butanediol with pyruvate decarboxylase from Candida tropicalis and method for the production of 2,3-butanediol therefrom}

The present invention introduces the pyruvate decarboxylase derived from Candida tropicallis, which is lower in activity than the self-retained pyruvate decarboxylase, into the cells, so that the production of ethanol acting as a by-product in the production of 2,3- However, the present invention relates to a recombinant yeast for producing 2,3-butanediol capable of producing acetyl-CoA required for growth, and a production method of 2,3-butanediol using the recombinant yeast.

2,3-butanediol is a chemical substance used in the synthesis of main constituents of solvents, anti-freeze agents, plasticizers, pharmaceuticals, and cosmetics. Methyl ethyl ketone (MEK) Is a biochemical material used as a precursor of 1,3-butadiene for the synthesis of styrene-butadiene rubber (SBR), a raw material for production and automobile tires.

In order to produce 2,3-butanediol using biological methods, it is necessary to develop a 2,3-butanediol strain and develop an optimal fermentation process by applying metabolic engineering techniques. In particular, it is necessary to develop a strain having high yield and high productivity for securing the safety of the strain for mass production of 2,3-butanediol and for commercialization.

On the other hand, 2,3-dimethyl butane as a conventional strain used for the production of all the keulrep when Ella oxy cytokine (Klebsiella oxytoca), Klebsiella pneumoniae (Klebsiella pneumoniae ), Aerobacter aerogenes , Bacillus subtilis , Paenibacillus < RTI ID = 0.0 > polymyxa , and Serratia marcescens .

However, although these strains can produce 2,3-butanediol with high yield / high productivity, they have been classified as pathogenic microorganisms and have inherent limitations in safety and industrialization. Thus, a technique for producing 2,3-butanediol using a yeast well known as a GRAS microorganism has been conventionally developed. However, yeast has the following limitations in industrial use as a 2,3-butanediol producing strain.

In the prior art, a yeast strain in which a core gene, PDC (pyruvate decarboxylase) gene, was removed was used to prevent conversion of pyruvate, which is a main precursor of 2,3-butanediol, to ethanol. This yeast strain succeeded in producing a high yield of 2,3-butanediol by introducing the 2,3-butanediol biosynthesis pathway, but cell growth and substrate consumption rate were remarkably decreased by removal of the PDC enzyme , The productivity of 3-butanediol was remarkably low.

Therefore, in order to commercially produce 2,3-butanediol using yeast, low cell growth rate and substrate consumption rate of existing strains have to be solved.

Korean Patent Publication No. 10-2015-0068581 (published on June 22, 2015) discloses that the biosynthesis pathway of 2,3-butanediol is 2 (2) with a recombinant yeast with metabolic engineering (3-butanediol), wherein pyruvate decarboxylase activity is inhibited and 2,3-butanediol biosynthesis-related foreign genes and xylose metabolism-related foreign genes are introduced Techniques for producing 2,3-butanediol from glucose or xylose using recombinant yeast are described.

In the present invention, a method for producing 2,3-butanediol with high productivity by solving a low cell growth rate and a substrate consumption rate of a conventional 2,3-butanediol producing strain is developed and provided.

The present invention relates to a process for the production of a protein which is transformed to lose the function of pyruvate decarboxylase and which is capable of producing Candida tropicallis is transformed to express pyruvate decarboxylase derived from tropicalis , is transformed to express acetolactate synthase, and acetolactate decarboxylase is expressed Butanediol dehydrogenase is transformed to produce a recombinant Saccharomyces cerevisiae for the production of 2,3-butanediol, which is characterized in that it is transformed so as to express butanediol dehydrogenase . At this time, pyruvate decarboxylase derived from Candida tropicalis may be composed of the amino acid sequence of SEQ ID NO: 2, for example.

The yeast strain for the production of 2,3-butanediol of the present invention can be obtained by introducing Candida tropicallis-derived Pdc, which is less active than its own pyruvate decarboxylase (Pdc), into the cells, Was able to increase the rate of growth of the cells and the rate of substrate consumption, and ultimately the productivity of 2,3-butanediol could be greatly improved.

On the other hand, in the recombinant Saccharomyces cerevisiae for the production of 2,3-butanediol of the present invention, the transformation of the pyruvate decarboxylase to function is lost, For example, access Celebi as Saccharomyces My jiae (Saccharomyces from cerevisiae) cells, pyruvate Cartesian acid la kinase 1 (pyruvate decarboxylase 1) of the crushing part of the PDC1 gene coding or removing the whole and, PDC5 gene coding for pyruvate Cartesian acid la kinase 5 (pyruvate decarboxylase 5) Can be accomplished by disrupting a portion of the PDC6 gene, removing the entire portion, and disrupting or completely removing a portion of the PDC6 gene encoding pyruvate decarboxylase 6.

The disappearance of the enzyme activity can be achieved by partially disrupting the gene encoding the enzyme or by removing the entire gene. Partial disruption and knock-out of the gene can be easily accomplished using techniques well known in the field of genetic engineering A detailed description thereof will be omitted.

On the other hand, in the recombinant Saccharomyces cerevisiae for producing 2,3-butanediol of the present invention, pyruvate decarboxylase derived from Candida tropicalis is expressed Transforming, for example, can be performed using Candida tropicallis can be accomplished by introducing the PDC1 gene encoding Saccharomyces cerevisiae , which encodes pyruvate decarboxylase 1 derived from tropicalis , into Saccharomyces cerevisiae cells. According to the experiments of the present invention described below, Candida tropicallis ( Candida The activity of the pyruvate decarboxylase 1 derived from tropicalis was suitably low, so that acetyl-CoA could be biosynthesized without producing ethanol, and cell growth and glucose consumption rate were high.

On the other hand, the trophy faecalis Candida (Candida in the present invention tropicalis) PDC1 gene of origin is preferably GPD2 (glyceraldehyde phosphate dehydrogenase 2) promoter. The GPD2 promoter may, for example, be composed of the amino acid sequence of SEQ ID NO: 2.

In addition, the Candida tropicallis tropicalis- derived PDC1 gene is preferably introduced into the cell by one copy. In the following experiment, when a single copy was introduced into the cells using the GPD2 promoter, cell growth and glucose consumption rate were high, ethanol was not produced, and ultimately productivity of 2,3-butanediol was high appear.

Meanwhile, the present invention provides a method for producing 2,3-butanediol, wherein the recombinant Saccharomyces cerevisiae of the present invention is cultured. At this time, the culture is preferably carried out using a medium containing glucose. Glucose is a preferred carbon source of Saccharomyces cerevisiae, and when it is used as a carbon source, the growth of the strain is maximized and the productivity can be increased.

Meanwhile, the method for producing 2,3-butanediol of the present invention is preferably performed while supplying oxygen, because a large amount of ATP is produced through oxygen supply, thereby promoting the growth of microorganisms.

On the other hand, the production method of 2,3-butanediol of the present invention can be carried out by fed-batch culture in which glucose is continuously supplied. By continuing to feed the medium in a fed-batch fashion, it is possible to produce 2,3-butanediol in one batch at maximum.

2,3-butanediol production technology using conventional yeast could not produce 2,3-butanediol with low productivity.

However, the yeast strain for the production of 2,3-butanediol of the present invention can be obtained by introducing Candida tropicallis-derived Pdc having lower activity than its own pyruvate decarboxylase (Pdc) into the cells, -CoA was able to increase the growth rate of the cells and the substrate consumption rate, and ultimately the productivity of 2,3-butanediol could be greatly improved.

That is, conventional Pdc-deficient strains did not produce acetyl-CoA in addition to ethanol biosynthesis in accordance with the lack of Pdc, so that the cells could not grow efficiently. In the present invention, this problem is solved by adding Pdc derived from Candida tropicallis into cells . Thus, the present invention was able to produce 2,3-butanediol of high concentration with high productivity.

1 (A) is a graph showing the result of fermentation with a 2,3-butanediol producing strain (control group) that does not express PDC . Fig. 1 (B) is a graph showing the fermentation result of the strain expressed by CtPDC1 . 1 (C) is a graph showing the fermentation result of the strain expressed by KmPDC1 .
Fig. 2 is a photograph of Candida tropicallis (promoter type, number of copies) of pyruvate decarvoxylase-expressing strains derived from P. tropicalis .
Figure 3 is a graph showing the yields for the products of the PDC expression strain (ethanol, glycerol, 2,3-butanediol) with various expression conditions.
4 (A) is a graph showing a result of fermentation of 2,3-butanediol in the control group for 120 hours. Fig. 4 (B) is a graph showing the results of fermentation of BD5_G1CtPDC1 strain for 120 hours of 2,3-butanediol.
FIG. 5 is a graph of 2,3-butanediol production results of BD5_G1CtPDC1 strain measured through fed-batch culture.

In the present invention, a 2,3-butanediol high productivity recombinant yeast was developed using a metabolic engineering technique, and 2,3-butanediol was produced from glucose with high productivity by using it .

In order to control the activity of Pdc encoding the pyruvate decarboxylase enzyme, a PDC gene with low activity was searched. Candida tropicallis (CtPDC1) gene derived from a tropicalis- derived nucleic acid sequence of SEQ ID NO: 1 and an amino acid sequence of SEQ ID NO: 2 to produce a single copy of the strain to produce 2,3-butanediol in high yield (See Reference 1 below).

[Reference Figure 1]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Manufacturing example  One: PDC1 , PDC5 , PDC6  Gene disruption and 2,3- Butanediol  The 2,3- Butanediol  Construction of production strain]

In the case of wild yeast, pyruvate produced from glucose metabolites is mostly converted to ethanol by pyruvate decarboxylase and alcohol dehydrogenase. Therefore, in order to produce 2,3-butanediol from yeast in high yield, it is necessary to prevent conversion of pyruvate to ethanol.

Therefore, in the present invention, the pyruvate decarboxylase genes PDC1 , PDC5 and PDC6 were first disrupted to construct a strain in which pyruvate decarboxylase activity was completely removed. In order to introduce the 2,3-butanediol biosynthetic pathway into this strain, acetolactate synthase ( alsS ) derived from Bacillus subtilis and acetolactate decarboxylase (acetolactate decarboxylase (alsD)) a saccharide in MRS celebrity bicyclic Ke was introduced into the plasmid containing the TDH3 promoter and the CYC1 terminator (S. cerevisiae), 2,3- butanediol to the Saccharomyces celebrity bicyclic Ke (S. cerevisiae) di Butanediol dehydrogenase ( BDH1 ) was introduced into a plasmid containing TDH3 promoter and CYC1 terminator (see, for example, Korean Patent Application Laid-Open No. 10-2015-0068581 filed by the present inventors) .

[ Example  1: low activity Pyruvate Decarboxylase ( pyruvate decarboxylase , PDC ) Search for genes]

(1) Overview

The productivity of 2,3-butanediol was low due to the low cell growth and substrate consumption rate of the yeast strain from which the PDC gene was removed.

Therefore, in order to efficiently produce 2,3-butanediol, the activity of pyruvate decarboxylase is appropriately regulated so as to secure a pyruvate substrate, which is a precursor of 2,3-butanediol, and simultaneously C2 In order to enable the supply of compounds, we sought to find low-activity PDC genes.

(2) Materials and methods

end. Genes and plasmids

Candida tropicallis tropicalis ), Cluj Vero My process Marcia Taunus (Kluyveromyces marxianus), Genomic DNA of Saccharomyces cerevisiae was extracted and PCR was carried out to obtain PDC1 (CtPDC1), Kluyveromyces marxianus ( Candida albicans) from Candida tropicalis the PDC1 (KmPDC1), three Levy jiae a saccharide My process (Saccharomyces cerevisiae) origin of PDC1 (ScPDC1), PDC5 (ScPDC5 ), PDC6 (ScPDC6) gene was cloned.

As the expression vector for yeast, the 'origin' of '2 micron plasmid' and the p426GPD plasmid containing TDH3 promoter and CYC1 terminator derived from S. cerevisiae were used.

The PDC gene fragment obtained by the PCR method was ligated to restriction sites of Xma I and Xho I of the p426GPD plasmid to construct a recombinant vector.

I. Yeast transformation

The thus-constructed recombinant vector was introduced into the PDC1 , 5, 6- removed yeast strain of Production Example 1 through transformation.

(3) Results

The kinase constant of 'K _m , V _max ' was calculated from activity values of various concentrations of substrate by measuring the 'in vitro enzyme activity' of the transformed yeast strain. The values are shown in Table 1 below.

Strain CtPDC1 KmPDC1 ScPDC1 ScpPDC5 ScPDC6 K _m (mM) 2.7 7.7 4.7 9.9 8.2 V _max (mU / mg protein) 107 383 541 437 415

As a result, the activity of CtPDC1 strain was significantly lower than that of other Pdc strains. Using a low activity CtPDC1 strain, it was determined that the Pdc activity control needed to optimize 2,3-butanediol production would be possible.

[ Example  2: CtPDC1 and KmPDC1 of  Cell growth, glucose consumption rate and 2,3-butanediol productivity of the expressed strain]

(1) Overview

Cell growth and glucose consumption rate were measured using the Pdc expression strain (CtPDC1, KmPDC1) prepared in Example 1, and the productivity of 2,3-butanediol was compared.

(2) Materials and methods

end. Strains and plasmids

2,3-butane diol-producing strain did not express Pdc, CtPDC1 expression strain, was used for expression KmPDC1 strain. CtPDC1 and KmPDC1 were expressed using the S. cerevisiae TDH3 promoter and the CYC1 terminator.

I. Medium and culture conditions

A medium containing 6.7 g / L of yeast nitrogen base w / o nitrogen base, 1.4 g / L of 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.

(3) Results

For non-expressing PDC control group, the glucose consumption rate whereas was 0.10 g / L / h per hour, the strain expressing the CtPDC1 is 0.94 g / L / h, the strain expressing the KmPDC1 is 1.09 g / L / h was .

Maximum dry cell concentration in the case of non-expressing PDC strains was 0.2 g _DCW / L, a strain expressing CtPDC1 is 2.7 g _DCW / L, a strain expressing KmPDC1 was 2.8 g _DCW / L.

PDC 2,3- butane diol which is not expressed strain and the ethanol yield was 0.259 g _{2,3- butane diol} / g _glucose and 0 g _ethanol / g _glucose were, a strain expressing CtPDC1 is 0.185 g _{2,3 -butane diol} / g _glucose, 0.185 g _ethanol / g _glucose, one strain expressing KmPDC1 is 0.134 g was _{2,3-butane diol} / g _glucose, 0.275 g _ethanol / g _glucose.

On the other hand, 2,3-butane diol productivity did not express PDC strains 0.026 g _2,3-butane was _{the yiol} / L / h, a strain expressing CtPDC1 is 0.175 g _{2,3-butane diol} / L / h, and the strain expressing KmPDC1 was 0.147 g _{2,3- butanediol} / L / h.

PDC from the above experiment - was confirmed that lacks 2,3-butane diol production results growth and glucose consumption rate significantly increased cell which expresses a PDC in strain, and confirmed that a significant increase in productivity (Figure 1). FIG. 1 (A) is a graph showing the result of fermentation with 2,3-butanediol producing strain (control group) that does not express PDC . FIG. 1 (B) is a graph showing the fermentation result of the strain expressed by CtPDC1 . 1 (C) is a graph showing the fermentation result of the strain expressed by KmPDC1 .

[ Example  3: Candida Trophy ( Candida tropicalis ) Origin Pyruvate Decar room Pyruvate decarboxylase ) ≪ / RTI > and < RTI ID = 0.0 > in vitro  activity assay ']

(1) Overview

Expression of PDC is therefore directly affects the yield and productivity of the all-2,3-dimethyl butane, the CtPDC1 expression strains with different expression conditions were constructed in order to choose the best CtPDC1 expression strains.

(2) Materials and methods

end. Plasmid construction with various promoters

Candida Tropical faecalis (C. tropicalis) amplifies the PDC1 gene (CtPDC1) derived from each of the expression vector-inserted (each CYC1 promoter as the promoter, the promoter sequence number GPD2 3-, CYC1 terminator both as a TDH3 promoter and terminator used) And then transformed into a recombinant yeast strain. The strain to be used for transformation was cultured in YNB medium for 2 days, and the cells at the time when the OD value became 3 were used.

I. Transformation

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

(3) Results

In order to confirm the degree of PDC expression of these strains, ' in vitro Pdc activity assay' was performed. As a result, the Pdc titer was not observed in the control group, and the constructed strain showed various Pdc expression levels (FIG. 2). FIG. 2 shows the result of measurement of the Pdc titer of a pyruvate decarvoxylase-expressing strain derived from Candida tropicalis .

[ Example  4: Candida Trophy ( Candida tropicalis ) Origin Pyruvate Decar room Production of 2,3-butanediol using a strain expressing pyruvate decarvoxylase]

(1) Overview

In order to confirm the fermentation behavior of the 2,3-butanediol of the strains constructed in Example 3 above, fermentation experiments were performed in a YNB medium containing 90 g / L of glucose at the initial stage. The control group was used as the transformed strain by "p426GPD ball CtPDC1 vector that does not contain".

(2) Fermentation method

The initial inoculum 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.

(3) Results

In the control group, 2,3-butanediol yield was 0.292 g _{2,3- butanediol} / g _glucose and no ethanol was produced.

A large amount of ethanol was produced in the BD5_C2CtPDC1 and BD5_T2CtPDC1 strains of the PDC expressing multiple copies of the PDC , and the yields of _{2,3- butanediol} were 0.245 and 0.150 g, respectively, _Lt; / RTI > _glucose (Fig. 3). Figure 3 is a graph showing the yields for the products of the control and PDC expression strains. 3, the production yields of 2,3-butanediol from BD5_C1CtPDC1 and BD5_G1CtPDC1 strains were similar, but BD5_C1CtPDC1 was not preferable due to the high production yield of glycerol as a by-product. Therefore, BD5_G1CtPDC1 strain was more suitable as a 2,3-butanediol producing strain.

On the other hand, the expression of PDC increased cell growth and glucose uptake rate. In the case of BD5_G1CtPDC1 strain, 0.62 g _glucose / L / h was observed in the control group, while the glucose consumption rate per unit time was 0.26 g _glucose / L / h in the control group. In the case of BD5_G1CtPDC1 strain, the yield of 2,3-butanediol production was similar to that of the control, but the productivity of 2,3-butanediol increased from 0.076 g / L / h to 0.181 g / L / h in the control group 4).

4 (A) is a graph showing the results of fermentation of 2,3-butanediol in the control group for 120 hours, and FIG. 4 (B) is a graph showing the results of fermentation of BD5_G1CtPDC1 strain for 2,3-butanediol for 120 hours.

[ Example 5: Oil price formula  The 2,3- Butanediol  production]

(1) Overview

In order to determine whether the strain BD5_G1CtPDC1 is suitable as a 2,3-butanediol producing strain, a fed-batch culture in which glucose was added in the middle of fermentation was carried out.

(2) Materials and methods

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

(3) Results

During the incubation time of 80 hours, 121.8 g / L of 2,3-butanediol was produced. At this time, the yield of _{2,3- butanediol} was 0.329 g, _{2,3- butanediol} / g _glucose , and the 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 strain BD5_G1CtPDC1.

These results indicate that the pyruvate decarboxylase-introduced strain derived from Candida tropicalis of the present invention is suitable for the production of 2,3-butanediol.

<110> SNU R & D FOUNDATION <120> Recombinant Saccharomyces cerevisiae for the production of          2,3-butanediol with pyruvate decarboxylase from Candida          tropicalis and method for the production of 2,3-butanediol          therefrom <130> AP-2015-0146 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1704 <212> DNA <213> Candida tropicalis <400> 1 atgtctgaaa ttactttggg tagattcttc tttgaaagat tgcaccaatt gcaagttgac 60 accgttttcg gtttaccagg tgattttaac ttggctttat tagataaaat ctacgaagtc 120 gatggtatga gatgggctgg taacgccaat gaattgaacg ctggttacgc tgctgatggt 180 tacgccagag ttaatccaaa tggtttggct gctttagtct ccaccttcgg tgttggtgaa 240 ttgtctttga ctaacgccat tgctggttct tactctgaac acgttggtat cattaacttg 300 gttggtgttc catcttcttc tgctcaagct aaacaattgt tgttgcacca caccttgggt 360 aacggtgatt tcactgtttt ccacagaatg ttcaagaaca tttctcaaac ttctgctttc 420 atctccgacc caaacactgc tgcttctgaa attgacagat gtatcagaga tgcttacgtt 480 taccaaagac cagtttacat tggtttgcca tctaacttgg ttgatgttaa agttccaaaa 540 tctttgttgg acaaaaaaat tgacttgtcc ttgcatccaa atgaaccaga atcccaagct 600 gaagttgttg aaaccgttga aaaattcatt tctgaagctt ctaacccagt tatcttggtt 660 gatgcttgtg ctatcagaca caactgtctt aaagaagttg ctgaattgat tgctgaaact 720 caattcccag tcttcaccac tccaatgggt aaatcaagtg ttgatgaatc caacccaaga 780 ttcggtggtg tttacgttgg ttctttgtct tctccagatg ttaaagaagc cgttgaaagt 840 gctgacttgg tcttatctgt tggtgctatg ttgtctgatt tcaacactgg tgctttctct 900 tacaactaca agaccagaaa tgttgttgaa ttccactctg attacaccaa gatcagacaa 960 gctactttcc caggtgtcca aatgaaagaa gctttgcaag ttttgttgaa gactgtcaag 1020 aaatctgtca atccaaaata cgtcccagct ccagttccag ctaccaaagc tattaccact 1080 ccaggtaaca acgacccagt ctctcaagaa tacttgtgga gaaaagtttc tgactggttc 1140 caagaaggtg atgttatcat ttctgaaacc ggtacctctg ctttcggtat tgtccaatct 1200 aaattcccaa agaatgccat tggtatttcc caagtcttgt ggggttctat tggttacgct 1260 actggtgcta cttgtggtgc tgctatggct gctcaagaaa ttgacccaaa gaagagagtt 1320 atcttgttca ctggtgatgg ttctttgcaa ttgactgtcc aagaaatctc taccatgtgt 1380 aaatgggatt gttacaacac ctatctttac gttttgaaca acgatggtta caccattgaa 1440 agattgattc acggtgaaaa agctcaatat aacgacattc aaccatggaa caacttgcaa 1500 cttttgccat tgttcaacgc taagaaatac gaaaccaaga gaatttctac tgttggtgaa 1560 ttgaacgatt tgttcactaa caaagaattt gctgttccag acagaattag aatggttgaa 1620 attatgttgc cagttatgga tgctccagct aacttggttg cccaagctaa acaatctgct 1680 gctaccaacg ctgctcaaga ataa 1704 <210> 2 <211> 567 <212> PRT <213> Candida tropicalis <400> 2 Met Ser Glu Ile Thr Leu Gly Arg Phe Phe Phe Glu Arg Leu His Gln   1 5 10 15 Leu Gln Val Asp Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ala              20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Asp Gly Met Arg Trp Ala Gly Asn          35 40 45 Ala Asn Glu Leu Asn Ala Gly Tyr Ala Ala Asp Gly Tyr Ala Arg Val      50 55 60 Asn Pro Asn Gly Leu Ala Ala Leu Val Ser Thr Phe Gly Val Gly Glu  65 70 75 80 Leu Ser Leu Thr Asn Ala Ile Ala Gly Ser Tyr Ser Glu His Val Gly                  85 90 95 Ile Ile Asn Leu Val Gly Val Ser Ser Ser Ala Gln Ala Lys Gln             100 105 110 Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His         115 120 125 Arg Met Phe Lys Asn Ile Ser Gln Thr Ser Ala Phe Ile Ser Asp Pro     130 135 140 Asn Thr Ala Ala Ser Glu Ile Asp Arg Cys Ile Arg Asp Ala Tyr Val 145 150 155 160 Tyr Gln Arg Pro Val Tyr Ile Gly Leu Pro Ser Asn Leu Val Asp Val                 165 170 175 Lys Val Pro Lys Ser Leu Leu Asp Lys Lys Ile Asp Leu Ser Leu His             180 185 190 Pro Asn Glu Pro Glu Ser Gln Ala Glu Val Val Glu Thr Val Glu Lys         195 200 205 Phe Ile Ser Glu Ala Ser Asn Pro Val Ile Leu Val Asp Ala Cys Ala     210 215 220 Ile Arg His Asn Cys Leu Lys Glu Val Ala Glu Leu Ile Ala Glu Thr 225 230 235 240 Gln Phe Pro Val Phe Thr Thr Pro Met Gly Lys Ser Ser Val Asp Glu                 245 250 255 Ser Asn Pro Arg Phe Gly Gly Val Tyr Val Gly Ser Leu Ser Ser Pro             260 265 270 Asp Val Lys Glu Ala Val Glu Ser Ala Asp Leu Val Leu Ser Val Gly         275 280 285 Ala Met Leu Ser Asp Phe Asn Thr Gly Ala Phe Ser Tyr Asn Tyr Lys     290 295 300 Thr Arg Asn Val Val Glu Phe His Ser Asp Tyr Thr Lys Ile Arg Gln 305 310 315 320 Ala Thr Phe Pro Gly Val Gln Met Lys Glu Ala Leu Gln Val Leu Leu                 325 330 335 Lys Thr Val Lys Lys Ser Val Asn Pro Lys Tyr Val Pro Ala Pro Val             340 345 350 Pro Ala Thr Lys Ala Ile Thr Thr Pro Gly Asn Asn Asp Pro Val Ser         355 360 365 Gln Glu Tyr Leu Trp Arg Lys Val Ser Asp Trp Phe Gln Glu Gly Asp     370 375 380 Val Ile Ile Ser Glu Thr Gly Thr Ser Ala Phe Gly Ile Val Gln Ser 385 390 395 400 Lys Phe Pro Lys Asn Ala Ile Gly Ile Ser Gln Val Leu Trp Gly Ser                 405 410 415 Ile Gly Tyr Ala Thr Gly Ala Thr Cys Gly             420 425 430 Glu Ile Asp Pro Lys Lys Arg Val Ile Leu Phe Thr Gly Asp Gly Ser         435 440 445 Leu Gln Leu Thr Val Gln Glu Ile Ser Thr Met Cys Lys Trp Asp Cys     450 455 460 Tyr Asn Thr Tyr Leu Tyr Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu 465 470 475 480 Arg Leu Ile His Gly Glu Lys Ala Gln Tyr Asn Asp Ile Gln Pro Trp                 485 490 495 Asn Asn Leu Gln Leu Leu Pro Leu Phe Asn Ala Lys Lys Tyr Glu Thr             500 505 510 Lys Arg Ile Ser Thr Val Gly Glu Leu Asn Asp Leu Phe Thr Asn Lys         515 520 525 Glu Phe Ala Val Pro Asp Arg Ile Arg Met Val Glu Ile Met Leu Pro     530 535 540 Val Met Asp Ala Pro Ala Asn Leu Val Ala Gln Ala Lys Gln Ser Ala 545 550 555 560 Ala Thr Asn Ala Ala Gln Glu                 565 <210> 3 <211> 1144 <212> DNA <213> Saccharomyces cerevisiae <400> 3 caaaaacgac atatctatta tagtggggag agtttcgtgc aaataacaga cgcagcagca 60 agtaactgtg acgatatcaa ctcttttttt attatgtaat aagcaaacaa gcacgaatgg 120 ggaaagccta tgtgcaatca ccaaggtcgt cccttttttc ccatttgcta atttagaatt 180 taaagaaacc aaaagaatga agaaagaaaa caaatactag ccctaaccct gacttcgttt 240 ctatgataat accctgcttt aatgaacggt atgccctagg gtatatctca ctctgtacgt 300 tacaaactcc ggttatttta tcggaacatc cgagcacccg cgccttcctc aacccaggca 360 ccgcccccag gtaaccgtgc gcgatgagct aatcctgagc catcacccac cccacccgtt 420 gatgacagca attcgggagg gcgaaaaata aaaactggag caaggaatta ccatcaccgt 480 caccatcacc atcatatcgc cttagcctct agccatagcc atcatgcaag cgtgtatctt 540 ctaagattca gtcatcatca ttaccgagtt tgttttcctt cacatgatga agaaggtttg 600 agtatgctcg aaacaataag acgacgatgg ctctgccatt gttatattac gcttttgcgg 660 cgaggtgccg atgggttgct gaggggaaga gtgtttagct tacggaccta ttgccattgt 720 tattccgatt aatctattgt tcagcagctc ttctctaccc tgtcattcta gtattttttt 780 tttttttttt tggttttact tttttttctt cttgcctttt tttcttgtta ctttttttct 840 agtttttttt ccttccacta agctttttcc ttgatttatc cttgggttct tctttctact 900 cctttagatt ttttttttat atattaattt ttaagtttat gtattttggt agattcaatt 960 ctctttccct ttccttttcc ttcgctcccc ttccttatca atgcttgctg tcagaagatt 1020 aacaagatac acattcctta agcgaacgca tccggtgtta tatactcgtc gtgcatataa 1080 aattttgcct tcaagatcta ctttcctaag aagatcatta ttacaaacac aactgcactc 1140 aaag 1144

Claims (9)

Is transformed to lose the function of pyruvate decarboxylase,
Candida tropicallis is transformed to express pyruvate decarboxylase derived from tropicalis ,
Is transformed to express acetolactate synthase,
Acetolactate decarboxylase is transformed to be expressed,
Butanediol dehydrogenase is expressed by the expression of the gene encoding Saccharomyces cerevisiae . The recombinant Saccharomyces cerevisiae for the production of 2,3-butanediol is characterized in that it is transformed to express butanediol dehydrogenase.
The method according to claim 1,
The transformation of the pyruvate decarboxylase to the disappeared function can be achieved by, for example,
Saccharomyces cerevisiae cells,
A part of the PDC1 gene encoding pyruvate decarboxylase 1 is disrupted or completely removed,
A part of the PDC5 gene encoding pyruvate decarboxylase 5 is disrupted or completely removed,
Butanediol is obtained by disrupting a part of the PDC6 gene coding for pyruvate decarboxylase 6 or by removing the entire part of the PDC6 gene encoding the pyruvate decarboxylase 6. The recombinant Saccharomyces cerevisiae Saccharomyces cerevisiae ).
The method according to claim 1,
Candida tropicallis Transformation of pyruvate decarboxylase derived from tropicalis to be expressed is,
Candida tropicallis which is characterized in that PDC1 gene coding for pyruvate decarboxylase 1 derived from tropicalis is introduced into Saccharomyces cerevisiae cells. Recombinant Saccharomyces cerevisiae for production ( Saccharomyces cerevisiae ).
The method of claim 3,
The Candida tropicallis ( Candida tropicalis- derived PDC1 gene,
Wherein the expression is regulated by a glyceraldehyde phosphate dehydrogenase 2 (GPD2) promoter. 2. The recombinant Saccharomyces cerevisiae for the production of 2,3-butanediol.
The method of claim 3,
The Candida tropicallis ( Candida tropicalis- derived PDC1 gene,
1 copies into the cells (copy) in 2,3-butane diol for the production of recombinant Saccharomyces characterized in that the introduction of my process three Levy jiae (Saccharomyces cerevisiae ).
The recombinant Saccharomyces cerevisiae of claim 1, cerevisiae ) is cultured.
The method according to claim 6,
The above-
Lt; RTI ID = 0.0 &gt; 2,3-butanediol &lt; / RTI &gt;
The method according to claim 6,
The above-
Butanediol is carried out while supplying oxygen.
The method according to claim 6,
The above-
A method for producing 2,3-butanediol characterized in that it is a fed-batch culture in which glucose is continuously supplied.

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