CN107937296B

CN107937296B - Recombinant saccharomyces cerevisiae with acetic acid, furfural and vanillin tolerance, and preparation method and application thereof

Info

Publication number: CN107937296B
Application number: CN201711229127.0A
Authority: CN
Inventors: 刘源涛; 孜力汗; 许建韧; 何艳艳
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2017-11-29
Filing date: 2017-11-29
Publication date: 2020-12-11
Anticipated expiration: 2037-11-29
Also published as: CN107937296A

Abstract

The invention relates to the technical field of microorganism biology, in particular to recombinant saccharomyces cerevisiae with furfural acetate vanillin tolerance, a preparation method and application thereof. The main technical scheme is as follows: a recombinant Saccharomyces cerevisiae with furfural acetate vanillin tolerance is named as S288C-YPL162C, the preservation number of the strain is CGMCC No.13925, the strain is preserved in the China general microbiological culture Collection center, and the preservation date is 2017, 03 and 23 days; the strain carries YPL162C gene, and the GenBank accession number of YPL162C gene is NM-001183976.1. The saccharomyces cerevisiae strain is unicellular fungus, and the shape of the saccharomyces cerevisiae strain is mostly oval and round. The colony morphology is similar to that of bacteria, but is larger and thicker, and is milky white, and the surface is wet and sticky. The saccharomyces cerevisiae S288C-YPL162C disclosed by the invention has good growth and ethanol fermentation performances under the environmental stress conditions of respectively containing high-concentration acetic acid, furfural, vanillin and the like.

Description

Recombinant saccharomyces cerevisiae with acetic acid, furfural and vanillin tolerance, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of microbial biology, and particularly relates to recombinant saccharomyces cerevisiae with acetic acid, furfural and vanillin tolerance, and a preparation method and application thereof.

Background

With the rapid development of economy, the problem of energy shortage is highlighted, and fuel ethanol is widely concerned as a clean renewable energy source. As a big agricultural country, the lignocellulose is rich in source, low in price and renewable. Therefore, the development of the method for producing the fuel ethanol by using the agricultural and forestry waste lignocellulose as the raw material has important significance. The lignocellulose is used as a substrate for producing fuel ethanol, firstly, the lignocellulose needs to be subjected to acid-base pretreatment, a large amount of toxic byproducts such as organic acids, furans, aromatic compounds and the like are generated in the process (Biotechnology for Biofuels, 2013, 6 (1): 1-10), and the normal operation of yeast ethanol fermentation is seriously influenced by inhibiting the yeast growth and ethanol production through interfering the cell membrane composition, influencing the intracellular enzyme activity and the like.

The research shows that the protein coded by YPL162C gene is located on the vacuolar membrane, the specific function is unknown, and the function is related to the cell cycle morphology (Genome Biology, 2010, 11: R24). Simultaneously participate in Zn²⁺Transport (Journal of Cell Biology, 2015, 211: 639-. Expression of YPL162C gene was up-regulated when Saccharomyces cerevisiae W303-1A was faced with oxidative stress, and it was possible that the protein encoded by YPL162C gene was involved in repair of Cell damage after oxidative stress (Molecular Biology of the Cell, 2006, 17: 1472-1482). However, the gene has less research on the tolerance of acetic acid, furfural and vanillin, and no report that the tolerance of acetic acid, furfural and vanillin can be improved by over-expression of the gene is found at present.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides the recombinant saccharomyces cerevisiae with acetic acid, furfural and vanillin tolerance, and the preparation method and application thereof.

The technical scheme of the invention is as follows: a recombinant Saccharomyces cerevisiae S288C-YPL162C (Saccharomyces cerevisiae) with acetic acid, furfural and vanillin tolerance is disclosed, the preservation number of the strain is CGMCC No.13925, the strain is preserved in the China general microbiological culture Collection center, and the preservation date is 2017, 03 and 23 days; the strain carries YPL162C gene, and the GenBank accession number of YPL162C gene is NM-001183976.1. The saccharomyces cerevisiae strain is unicellular fungus, and the shape of the saccharomyces cerevisiae strain is mostly oval and round. The colony morphology is similar to that of bacteria, but is larger and thicker, and is milky white, and the surface is wet and sticky. The nutrition mode is heterotrophic facultative anaerobic type. The product is free and milky white when growing in the culture medium.

The GenBank accession number of the PGK1 promoter sequence is FJ415226.1, and the GenBank accession number of the CYC1 terminator sequence is EF 210198.1. A pHO constitutive expression vector is formed by connecting a PGK1 strong promoter and a CYC1 terminator on the basis of an pHO integration vector (NCBI: # AF324728, David J.Stillman benefit of Utah university, USA; Nucleic acids research, 2001, 29: e59) of yeast through a genetic engineering means. Earlier studies showed that self-flocculating yeast SPSC01 and free yeast SPSC01_△FLO1Compared with the resistance to environmental stress, the YPL162C gene is obtained by PCR amplification by using a self-flocculating yeast SPSC01 genome as a template and is connected between a PGK1 promoter and a CYC1 terminator. After linearization, the DNA was introduced into Saccharomyces cerevisiae S288C by chemical transformation method for overexpression (Nature Protocols, 2007, 2(1): 31-35). The introduced target fragment is integrated and expressed at the HO gene site of the Saccharomyces cerevisiae genome (Yeast, 1997, 13: 1563-1573).

The invention adopts pHO constitutive expression vector of saccharomyces cerevisiae to carry out integration expression of recombinant strain, pHO constitutive expression vector can carry out integration expression at HO gene site of saccharomyces cerevisiae in a homologous recombination mode, the vector inserts PGK1 promoter and CYC1 terminator at the multiple cloning site of plasmid by a genetic engineering means to obtain constitutive integration expression vector, and screening is carried out according to the geneticin G418 resistance contained in the vector (screening is carried out on a plate containing G418, only an individual successfully introduced with a target fragment containing G418 gene can grow on the plate).

The invention also claims the application of the saccharomyces cerevisiae S288C-YPL162C under the condition of inhibitor stress, such as good growth under the condition of high concentration of acetic acid, furfural or vanillin and higher ethanol fermentation efficiency compared with the control strain S288C-HO.

The invention has the following beneficial effects: (1) the invention provides a novel strain; (2) the recombinant saccharomyces cerevisiae S288C-YPL162C can grow well and ferment ethanol under the environmental stress conditions of respectively containing high-concentration acetic acid, furfural, vanillin and the like.

Drawings

FIG. 1 PCR amplification of YPL162C gene fragment;

FIG. 2 YPL162C vector;

FIG. 3 PCR identification of positive transformants;

FIG. 4 is a comparison of expression levels of YPL162C gene in recombinant Saccharomyces cerevisiae S288C-YPL162C and recombinant Saccharomyces cerevisiae S288C-HO control strain;

FIG. 5 microscopic examination of recombinant unloaded yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL 162C;

wherein: (a) S288C-HO, (b) S288C-YPL 162C.

FIG. 6 comparison of the growth of recombinant unloaded yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL162C in plates containing different inhibitors;

wherein: (a) growth of the over-expressing strain and the control strain without addition of inhibitor; (b) the growth conditions of the over-expression strain and the control strain under the addition of 10g/L acetic acid; (c) the growth conditions of the over-expression strain and the control strain under the condition of adding 1g/L of vanillin; (d) growth of the over-expression strain and the control strain with 1.5g/L furfural addition.

FIG. 7 recombinant unloaded yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL162C ethanol fermentation with 10g/L acetic acid addition;

wherein: (a) growth of the over-expressing strain and the control strain with the addition of 10g/L acetic acid as a function of time; (b) the ethanol concentration and the residual glucose in the fermentation liquid changed with time under the addition of 10g/L acetic acid.

FIG. 8 recombinant unloaded yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL162C ethanol fermentation with 2g/L vanillin addition;

wherein: (a) growth of the over-expressing strain and the control strain with the addition of 2g/L vanillin as a function of time; (b) the ethanol concentration and the residual glucose in the fermentation liquid change with time under the addition of 2g/L vanillin.

FIG. 9 recombinant unloaded yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL162C ethanol fermentation with 3g/L furfural addition;

wherein: (a) the growth of the over-expression strain and the control strain changes along with time under the condition of 3g/L furfural addition; (b) the change of the ethanol concentration and the residual glucose in the fermentation liquid with the addition of 3g/L furfural along with the time.

Detailed Description

Example 1: construction and transformation of recombinant Saccharomyces cerevisiae of unknown functional protein gene YPL162C

The YPL162C gene is obtained from NCBI public database, and the GenBank accession number of YPL162C gene is NM-001183976.1. The promoter of the gene is PGK1 promoter, CYC1 terminator is used as terminator, and YPL162C gene is inserted between PGK1 promoter and CYC1 terminator by enzyme digestion connection. The constructed plasmid is then cut linearly by restriction endonuclease Not I, and transformed into Saccharomyces cerevisiae S288C for integration and expression.

1.1 Saccharomyces cerevisiae genomic DNA extraction

(1) Single colonies of yeast were picked from freshly grown YPD plates, inoculated into 100mL of liquid YPD medium, and cultured overnight at 30 ℃ and 150 rpm.

(2) Taking 4mL of bacterial liquid, centrifuging at 10000rpm for 1min, collecting yeast cells, washing with deionized water for 2 times, and removing impurities on the cell surfaces such as a culture medium.

(3) To the yeast cells, 500. mu.L of a lysis solution (2% Triton X-100, 0.1M Tris. HCl pH 8.0, 50mM EDTA, 2% SDS) was added, and the yeast cells were resuspended and transferred to a 1.5mL centrifuge tube.

(4) Approximately 500. mu.L of acid-washed glass beads 0.5mM in diameter were added, mixed with 25. mu.L of a 5mM NaCl solution, vortexed at high speed for 5min, and the cells were disrupted.

(5) The cell disruption solution was centrifuged at 12000rpm for 5min, and the supernatant was transferred to a clean 1.5mL centrifuge tube.

(6) Adding equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) into the supernatant obtained after the centrifugation in the step (5), shaking and mixing uniformly, centrifuging at 12000rpm for 5min, and transferring the supernatant into a clean centrifuge tube.

(7) Adding 2 times volume of precooled absolute ethyl alcohol into the supernatant obtained after the centrifugation in the step (6), standing for 30min at the temperature of minus 80 ℃, precipitating the genome DNA, and centrifuging for 10min at the temperature of 4 ℃ and 12000 rpm.

(8) The supernatant was discarded, the pellet was washed with pre-cooled 70% ethanol 2 times, left at room temperature for 5min to volatilize ethanol, and the DNA pellet was dissolved in 50. mu.L of TE buffer.

(9) Add 1. mu.L 10mg/mL RNase A, incubate at 37 ℃ for 30min, digest RNA, store at-20 ℃.

1.2PCR amplification of the Gene band of interest

The gene YPL162C was amplified using the flocculated yeast SPSC01 DNA as a template. The primer sequences are as follows:

the PCR reaction was as follows (50. mu.L):

setting a PCR reaction program:

after the PCR was completed, the product was subjected to agarose gel electrophoresis to detect the fragment size, which was consistent with the sequence length in the NCBI database (FIG. 1).

1.3 cleavage of the target fragment

The PCR product was purified using a DNA purification kit and then cleaved with an enzyme. The enzyme digestion reaction system is as follows:

the reaction was incubated at 37 ℃ for 3 h.

And recovering the target DNA fragment by using a gel recovery kit, and freezing and storing at-20 ℃ for later use after detection.

1.4 connection of the Gene of interest to the vector (25. mu.L)

And connecting the prepared target fragment with the vector fragment after enzyme digestion and recovery.

The reaction system is as follows:

the reaction time was 10h in the ligator and the reaction temperature was 16 deg.C (see FIG. 2 for the constructed vector).

1.5 ligation product transformation of E.coli DH 5. alpha

1.5.1 preparation of competent cells of Escherichia coli

(1) Inoculating 200. mu.L of Escherichia coli DH5 stored at-80 deg.C into LB liquid culture medium, culturing at 37 deg.C and 200rpm overnight;

(2) transferring overnight cultured bacterial liquid into 50mL fresh LB liquid culture medium at a ratio of 1:100, culturing at 37 deg.C and 200rpm for 3-4h to OD_600nmAbout 0.5;

(3) transferring the bacterial liquid into a 50mL centrifugal tube precooled on ice, placing the centrifugal tube on ice for 30min, and centrifuging the centrifugal tube at 4 ℃ and 4000rpm for 5 min;

(4) discarding the supernatant, using pre-cooled 0.1mol/L CaCl₂15mL of suspension cells are placed on ice for 30min, centrifuged at 4 ℃ and 4000rpm for 5min, and the supernatant is discarded;

(5) repeating the above steps;

(6) adding pre-cooled 0.1mol/L CaCl₂Resuspend cells in 2mL of solution, add 2mL of pre-cooled 30% glycerol, mix gently, and split into 200. mu.L aliquots and freeze-store at-80 ℃.

1.5.2 transformation of ligation products

(1) Taking 200 μ L of competent cells from a refrigerator at-80 deg.C, and thawing on ice;

(2) adding 10 μ L of the ligation product into the competent cells, gently mixing with a pipette, and standing on ice for 30 min;

(3) placing the competent cells in a water bath kettle at 42 ℃ for 90s by heat shock, and then rapidly placing the competent cells in an ice bath for cooling for 2 min;

(4) adding 800 μ L LB liquid culture medium (without antibiotic) into the tube, mixing, and performing shaking culture at 37 deg.C for 1 h;

(5) centrifuging at 4000rpm for 5min, discarding part of the supernatant, mixing the rest solution, spreading on a screening plate containing corresponding antibiotics (final concentration of ampicillin is 100 μ g/mL), standing for half an hour with the front side facing upwards, air drying the bacteria solution completely, inverting the culture dish, and culturing at 37 deg.C for 12-16 h;

(6) and selecting positive colonies for identification.

1.5.3 transformant plasmid extraction

Transformant plasmids (E.Z.N.A Plasmid DNA MiNi Kit I Protocol-Spin Protocol) were extracted using an OMEG Plasmid extraction Kit.

1.5.4 PCR identification of Positive transformants

The extracted plasmid was used as a template for PCR reaction to verify the transformant. The target gene was amplified according to the method of 1.2 and verified by PCR.

After the PCR is finished, the product is subjected to agarose gel electrophoresis to detect whether the size of the gene fragment is correct or not.

1.6 linearization of recombinant plasmids

Since the recombinant plasmid needs to be linearized and then transferred into yeast for integration and expression, the plasmid is digested with Not I restriction enzyme. The enzyme digestion reaction system is as follows:

the reaction was incubated at 37 ℃ for 10 h.

And recovering the target DNA fragment by using a gel recovery kit, and freezing and storing at-20 ℃ for later use after detecting without errors.

Example 2: saccharomyces cerevisiae chemical transformation of unknown protein gene YPL162C

The saccharomyces cerevisiae cell chemical transformation method comprises the following steps:

(1) inoculating yeast strain S288C preserved at-80 deg.C into YPD culture medium, culturing at 30 deg.C and 150rpm for 12-14 hr, and inoculating into new YPD culture medium (1% inoculation) for 6 hr;

(2) centrifuging with a 50mL centrifuge tube to collect thalli, and centrifuging at 3000rpm for 3 min;

(3) discarding the supernatant, resuspending the cells with 20mL of sterile ultrapure water, and centrifuging at 3000rpm for 3 min;

(4) discarding the supernatant, resuspending the cells with 20mL of 0.1M LiAC (lithium acetate solution), and centrifuging at 3000rpm for 3 min;

(5) discard the supernatant, resuspend the cells with 0.5-2mL of 0.1M LiAC to OD_620nm2.0-3.0;

(6) taking 100 mu L of the bacterial liquid into a 1.5mL centrifuge tube, and centrifuging for 3min at 3000 rpm;

(7) discarding the supernatant, adding 240 mu L of 50% PEG (average molecular weight 3350-4000), 36 mu L of 1M LiAC, 50 mu L of 2mg/mL single-stranded salmon sperm DNA and 1 mu g of DNA fragment into a centrifuge tube, and uniformly mixing;

(8) water bath at 42 ℃ for 40-60 min;

(9) centrifuging at 3000rpm for 3min, discarding supernatant, adding 800 μ L YPD medium, culturing at 30 deg.C and 150rpm for 1-2 hr;

(10) centrifuging at 3000rpm for 3min, discarding part of supernatant to obtain concentrated bacterial liquid, and coating on YPD plate containing G418 300 μ G/mL;

(11) the plates were placed upside down in an incubator at 30 ℃ until yeast transformant colonies grew.

(12) Single colonies with good growth status were randomly picked and inoculated into YPD liquid medium (containing 100. mu.g/mL of G418 antibiotic), a certain amount of cells were obtained and subjected to genomic DNA extraction (method same as 1.1), and a verification fragment (primers shown below) was amplified using the obtained genome as a template to verify (method same as 1.2) the integration of the target gene, the verification fragment comprising a part of the target gene YPL162C fragment and a part of the resistance gene fragment G418, and the total length was 1000 bp. And further verified by agarose gel electrophoresis (fig. 3).

The verification fragment primers were as follows:

example 3: expression of YPL162C gene in recombinant strain S288C-YPL162C and microscopic detection of cell growth morphology

3.1 expression of the recombinant YPL162C Gene

(1) Inoculating the recombinant unloaded yeast S288C-HO control strain and the recombinant saccharomyces cerevisiae S288C-YPL162C into 100mL of seed culture medium (30g/L glucose, 3g/L peptone, 4g/L yeast extract powder), culturing at 30 ℃ and 150rpm for 24 h;

(2) respectively taking bacterial liquid, measuring light absorption value at 620nm, and regulating OD with seed culture medium₆₂₀To 1.5;

(3) inoculating 10% (v/v) of the culture medium (100g/L glucose, 3g/L peptone, 4g/L yeast extract powder, pH 4.5) to the fermentation medium, and culturing at 30 deg.C and 150 rpm;

(4) fermenting the strain to 12h (logarithmic phase), respectively taking 4mL of bacterial liquid, centrifuging, removing supernatant, washing cells for 2 times by using sterile water, quickly freezing thalli by using liquid nitrogen, preserving at-80 ℃, and extracting RNA for later use;

(5) extracting total RNA of recombinant bacteria S288C-YPL162C and control bacteria S288C-HO logarithmic growth phase by adopting a radix RNAsimple total RNA extraction kit;

(6) the extracted RNA is reversely transcribed into cDNA by referring to a TaKaRa PrimeScript TM RT reagent Kit with gDNA Eraser Kit method; Real-Time PCR reference

Premix Ex TaqTM II kit method was performed, and the obtained data were analyzed for the relative expression amount of the gene.

As a result: the expression level of the recombinant strain S288C-YPL162C gene YPL162C was 2.4 times that of the control strain without adding any inhibitor. It was revealed that the desired gene YPL162C was overexpressed in the recombinant bacteria, and the results are shown in FIG. 4.

3.2 detection of cell growth morphology by recombinant Strain microscopy

(1) Inoculating recombinant unloaded yeast S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL162C into 100mL seed culture medium (30g/L glucose, 3g/L peptone, 4g/L yeast extract), culturing at 30 deg.C and 150rpm to middle logarithmic phase (12 h);

(2) 1. mu.L of cells in the logarithmic growth phase of S288C-HO and S288C-YPL162C, respectively, were taken, and cell sections were prepared, observed with a microscope for cell morphology, and photographed.

As a result: as shown in FIG. 5, the S288C-HO control strain and the recombinant Saccharomyces cerevisiae S288C-YPL162C have no obvious difference in cell morphology observed under a microscope, which indicates that the cell morphology of the original strain is not affected after the YPL162C gene is over-expressed.

Example 4: comparison of growth of plates of recombinant Saccharomyces cerevisiae S288C-HO control strain and recombinant Saccharomyces cerevisiae S288C-YPL162C under different stress factors

(1) Inoculating the recombinant unloaded yeast S288C-HO control strain and the recombinant saccharomyces cerevisiae S288C-YPL162C into 100mL of seed culture medium (30g/L glucose, 3g/L peptone, 4g/L yeast extract powder), culturing at 30 ℃, 150rpm and over night;

(2) respectively inoculating overnight-cultured bacterial liquid into seed culture medium with an inoculum size of 10% (v/v) (same as step (1)), and culturing at 30 deg.C and 150rpm for 12 h;

(3) respectively taking bacterial liquid, measuring the light absorption value of the bacterial liquid at 620nm, and adjusting the OD value to 1.0 by using a sterilized seed culture medium;

(4) respectively carrying out 5-time gradient dilution on the bacterial liquid;

(5) sampling 2 μ L of the bacteria solution after gradient dilution in turn on YPD plates (20g/L glucose, 20g/L peptone, 10g/L yeast extract powder, 15g/L agar powder) containing 10g/L acetic acid, 1.5g/L furfural, 1g/L vanillin and no inhibitor;

(6) and (5) carrying out inverted culture in an incubator at 30 ℃, observing and comparing after bacterial colonies grow out, and photographing.

As a result: as seen from FIG. 6(a) control group, the growth status of the two strains S288C-HO and S288C-YPL162C were consistent under normal culture conditions (36 h of culture), indicating that YPL162C gene had no effect on the growth of the strains after overexpression.

As shown in FIG. 6(b), when 10g/L acetic acid was added to the medium (culture for 36 hours), the rest was addedUnder constant conditions, strain S288C-YPL162C was found to be 5^-6The dilution gradient had colony growth, while the control strain S288C-HO was at 5^-5The dilution gradient can not grow basically, which shows that the overexpression of the YPL162C gene improves the acetic acid tolerance of the recombinant strain S288C-YPL 162C.

Also as shown in FIGS. 6(c) and (b), when 1.5g/L furfural and 1g/L vanillin are added into the culture medium respectively, the growth state of the S288C-YPL162C strain in the solid culture medium is obviously better than that of the control strain S288C-HO, which indicates that the tolerance of the strain to furfural and vanillin is improved by over-expressing the YPL162C gene.

The plate growth experiments under different stress conditions show that after an unknown functional protein gene YPL162C is integrated and expressed on the chromosome of the saccharomyces cerevisiae S288C, the obtained saccharomyces cerevisiae strains S288C-YPL162C respectively have obviously enhanced tolerance to inhibitors of acetic acid, furfural and vanillin.

Example 5: comparison of ethanol fermentation conditions of recombinant Idle Yeast S288C-HO control Strain and recombinant Saccharomyces cerevisiae S288C-YPL162C with the addition of different inhibitors

(1) Respectively inoculating a control strain S288C-HO and a recombinant saccharomyces cerevisiae S288C-YPL162C into a 250mL shake flask containing 100mL of seed culture medium (30g/L glucose, 3g/L peptone and 4g/L yeast extract powder), and culturing at 30 ℃ and 150rpm for 24 h;

(2) respectively taking bacterial liquid, measuring the light absorption value of the bacterial liquid at 620nm, and then adjusting the OD to 1.5 by using a seed culture medium;

(3) the cells were inoculated into a fermentation medium containing 10g/L acetic acid, 2g/L vanillin and 3g/L furfural (100g/L glucose, 3g/L peptone, 4g/L yeast extract powder, pH 4.5) at an inoculum size of 10% (v/v), respectively, and subjected to ethanol fermentation at 30 ℃ and 150 rpm.

(4) Sampling at fixed time in the ethanol fermentation process of unloaded yeast S288C-HO and recombinant saccharomyces cerevisiae S288C-YPL162C, and respectively taking a certain amount of bacterial liquid to determine the density of the thallus by using an enzyme-linked immunosorbent assay to measure the light absorption value; and centrifuging the residual bacterial liquid, taking supernatant, detecting the concentration of residual glucose in the fermentation liquor by a DNS method, detecting the concentration of ethanol in the fermentation liquor by a gas chromatograph, and determining that the ethanol fermentation is finished when the concentration of the glucose in the supernatant is lower than 1 g/L.

As a result: as shown in FIG. 7(a), the over-expression strain S288C-YPL162C and the control strain S288C-HO enter the log phase at the same time in 12h with the progress of ethanol fermentation, the two strains reach the maximum value at the same time in 48h of fermentation, and the recombinant strain has a cell density 9% higher than that of the control strain. From FIG. 7(b), it is seen that the glucose consumption rate and ethanol production rate of the over-expressed strain S288C-YPL162C were both higher than those of the control strain S288C-HO. At 36h of fermentation, S288C-YPL162C had consumed all glucose, 12h earlier than the control strain S288C-HO. At 36h, the ethanol concentration of the recombinant strain reaches the maximum value of 37.3g/L, the ethanol concentration of the control strain reaches the maximum value of 36.8g/L at 48h, and the fermentation time is advanced by 12 h. The over-expression strain S288C-YPL162C is shown to have better growth state and fermentation performance than S288C-HO in a fermentation medium containing 10g/L of acetic acid, and the acetic acid tolerance of the strain is improved.

As seen from FIG. 8(a), during the ethanol fermentation process, the OD value of the over-expression strain S288C-YPL162C rapidly increased from 12h to 60h, and the cell density reached the maximum value 6h earlier than that of the control strain, and was increased by 71.4% compared with that of the control strain. As shown in FIG. 8(b), the glucose consumption rate and ethanol production rate of the over-expressed strain S288C-YPL162C were higher than those of the control strain S288C-HO, and the glucose consumption was completed and the ethanol concentration reached the maximum at 36h, which was 44h earlier than that of the control strain. The growth state and fermentation performance of the recombinant saccharomyces cerevisiae S288C-YPL162C with the over-expression gene YPL162C in a fermentation medium containing 2g/L of vanillin are obviously superior to those of S288C-HO, and the tolerance of the strain to the vanillin is enhanced.

As seen from FIG. 9(a), S288C-YPL162C entered the logarithmic phase 30h earlier than the control strain, and the maximum cell density was increased by 52% as compared with the control strain. As shown in FIG. 9(b), the strain S288C-YPL162C entered the rapid glucose consumption stage from 12 hours and consumed glucose 44 hours earlier than the control strain. The strain S288C-YPL162C has ethanol concentration up to 36g/L in 42h, the maximum ethanol concentration is increased by 24% compared with that of a control bacterium, and the fermentation time is shortened by 38 h. Therefore, the growth state and fermentation performance of the recombinant strain S288C-YPL162C in 3g/L furfural fermentation are obviously superior to those of a control strain, and the furfural tolerance of the original strain is obviously improved.

Claims

1. A recombinant saccharomyces cerevisiae with acetic acid, furfural and vanillin tolerance is characterized by being named as S288C-YPL162C, having a preservation number of CGMCC No.13925, being preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and having a preservation date of 2017, 03 and 23 days; the recombinant saccharomyces cerevisiae carries YPL162C gene, and the GenBank accession number of the gene is NM-001183976.1.

2. A method for constructing the recombinant Saccharomyces cerevisiae having acetic acid, furfural and vanillin tolerance of claim 1, comprising the steps of: the YPL162C gene obtained by PCR amplification is connected to a pHO constitutive integration expression vector by taking a self-flocculating yeast SPSC01 genome as a template, and is transferred into saccharomyces cerevisiae S288C after linearization to realize integration expression.

3. A method for constructing recombinant Saccharomyces cerevisiae having acetic acid, furfural and vanillin tolerance according to claim 1, comprising the steps of:

s1 construction and transformation of recombinant Saccharomyces cerevisiae containing YPL162C gene;

s1.1, extracting Saccharomyces cerevisiae genome DNA;

S1.2PCR amplifying target gene band;

s1.3, enzyme digestion of a target fragment;

s1.4 connecting the target gene with a vector;

s1.5 the ligation product is transformed into Escherichia coli DH5 alpha;

s1.6 linearization of recombinant plasmid;

and (3) obtaining a transformant by a S2 saccharomyces cerevisiae chemical transformation method.

4. The use of the recombinant Saccharomyces cerevisiae having acetic acid, furfural, and vanillin tolerance of claim 1 under inhibitor stress conditions.

5. The use of claim 4, wherein the inhibitor stress condition is a high concentration of acetic acid, a high concentration of furfural or a high concentration of vanillin.