CN113106029A

CN113106029A - Saccharomyces cerevisiae engineering bacterium for over-expressing ARO8 gene and construction method and application thereof

Info

Publication number: CN113106029A
Application number: CN202110423676.1A
Authority: CN
Inventors: 应汉杰; 朱家庆; 张涛; 赵伟; 蒋颖; 王诗萌; 张德力; 陈勇; 柳东; 余斌
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2021-07-13

Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for over-expressing an ARO8 gene, and a construction method and application thereof, wherein the saccharomyces cerevisiae engineering bacterium introduces a recombinant plasmid carrying an ARO8 gene into original saccharomyces cerevisiae. According to the invention, the saccharomyces cerevisiae engineering bacteria for over-expressing the ARO8 saccharomyces cerevisiae gene are constructed, so that the cell permeability of the bacteria is improved, and the yield of the biofilm is improved, thereby improving the ethanol yield of the saccharomyces cerevisiae in the fermentation process, increasing the colony number and the adhesion, improving the sugar conversion rate and the ethanol yield, and shortening the fermentation period. According to the invention, the saccharifying enzyme preparation and the saccharomyces cerevisiae cells are co-immobilized by an embedding method, and on the basis of treatment of the permeability of cell membranes of the saccharomyces cerevisiae, the biomembrane yield and the glucose conversion rate of the saccharomyces cerevisiae ARO8 gene overexpression genetic engineering bacteria are further improved, the fermentation period is shortened, and the sugar consumption is reduced.

Description

Saccharomyces cerevisiae engineering bacterium for over-expressing ARO8 gene and construction method and application thereof

Technical Field

The invention relates to the field of fermentation, in particular to saccharomyces cerevisiae engineering bacteria for over-expressing ARO8 gene, a construction method and application thereof.

Background

Biofilm, also called biofilm, is a multicellular population formed spontaneously by microorganisms and fixed on the surface of a medium, and the main components of the biofilm are microbial cells and Extracellular Polymers (EPS) secreted by the microbial cells, so that the biofilm is concerned about the survival of the cell population because the biofilm can increase the stress resistance of the cell population. The growth of the biofilm is a non-static process and is mainly divided into five periods, namely an initial adhesion period, an irreversible fixation period, a growth period, a maturation period and a diffusion period. The extracellular secretion forms a relatively stable structure with a cell colony with the increase of time, and the stress resistance of the colony is greatly enhanced. In the fermentation process of producing ethanol by saccharomyces cerevisiae, one of the important factors limiting the yield of ethanol is the tolerance of substrates and products, and high concentration of sugar and ethanol can generate toxic effect on the yeast and influence the normal growth and fermentation of cells. Generally, sugar exceeding 200g/L will have a deleterious effect on Saccharomyces cerevisiae cells, whereas glucose tolerance of yeast can be up to 300g/L or more by immobilized fermentation. The immobilized fermentation can also improve the continuity and fermentation efficiency of the saccharomyces cerevisiae, 50g/L of glucose can be consumed in 8h of the conventional free fermentation as a carbon source, the immobilized fermentation saccharomyces cerevisiae only needs about 6h for consuming the same glucose, and the whole immobilized fermentation system can enter a relatively stable state along with the increase of fermentation batches, and at the moment, the whole system can stably perform ethanol fermentation of dozens of batches in a fermentation period of 4 h. Therefore, how to regulate the function of the biofilm in the immobilized fermentation is important. Biofilms have been shown to have a positive role in fermentation production, and both biofilm size and membrane thickness are important influencing factors in microbial fermentation. Too much biofilm may cause difficulty in contacting the cells with nutrients, accumulation of metabolic waste products and damage to the cells themselves, affecting fermentation yield; and the fermentation advantage is not obvious when the biofilm is too little.

The permeability of the cell membrane is one of the characteristics of the cell membrane, and can help the cell to retain or discharge certain substances, and the permeability of the cell membrane can be changed by artificially treating the cell, so that the discharge efficiency of metabolites and the substance exchange of the cell are changed. The formation of the biofilm is closely related to the material exchange and metabolism of cells, and the change of the permeability of cell membranes can greatly improve the yield of the biofilm.

Embedding immobilization is a technology for simultaneously immobilizing enzyme and enzyme, enzyme and cell, and cell in vivo to form an immobilization system, and is the most widely applied immobilization method in saccharomyces cerevisiae at present. The embedding method is mainly classified into a natural carrier embedding method and an organic synthetic carrier embedding method. The natural carrier embedding method mainly comprises agar, carrageenan, calcium alginate, carrageenan, sodium alginate, polyvinyl alcohol and the like. They are characterized by biological non-toxicity, convenient molding and high immobilization density, but low mechanical strength, weak mass transfer capability and poor antimicrobial decomposition capability. The polyacrylamide, polyacrylic acid and polyvinyl alcohol used in the organic synthesis carrier embedding method have stable chemical properties and high mechanical strength, but the polymer forming conditions are severe in the immobilization process, so that the cells are damaged. In addition, since cells are embedded in the carrier, there are problems of poor mass transfer, cell death in the later stage, and low catalytic efficiency.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a saccharomyces cerevisiae engineering bacterium aiming at the defects of the prior art.

The invention also aims to solve the technical problem of providing a construction method of the saccharomyces cerevisiae engineering bacteria.

The invention further aims to solve the technical problem of providing the application of the saccharomyces cerevisiae engineering bacteria.

In order to solve the first technical problem, the invention discloses a saccharomyces cerevisiae engineering bacterium, wherein an ARO8 gene in the strain is over-expressed, namely, a recombinant plasmid carrying an ARO8 gene is introduced into original saccharomyces cerevisiae.

Wherein before the ARO8 is over-expressed, the ARO8 gene sequence is shown as SEQ ID NO: 1, after the ARO8 is over-expressed, the gene sequence of the recombinant plasmid carrying the ARO8 gene is shown as SEQ ID NO: 3, respectively.

Wherein the original Saccharomyces cerevisiae S288 c.

Wherein the recombinant plasmid carrying the ARO8 gene is a PYX212 recombinant plasmid carrying an ARO8 gene.

In order to solve the second technical problem, the invention discloses a construction method of the saccharomyces cerevisiae engineering bacteria, which comprises the following steps:

(1) using the genome of the original saccharomyces cerevisiae as a template, and carrying out PCR amplification on an ARO8 gene;

(2) inserting the ARO8 gene obtained in the step (1) into the NHE1 enzyme cutting site of the PYX212 plasmid to obtain the PYX212 recombinant plasmid carrying the ARO8 gene;

(3) transforming the recombinant plasmid obtained in the step (2) into a competent cell of E.coli DH5 alpha, and extracting the recombinant plasmid from E.coli DH5 alpha;

(4) and (4) transforming the recombinant plasmid extracted in the step (3) into competent cells of the original saccharomyces cerevisiae to obtain the saccharomyces cerevisiae engineering bacteria.

In the step (1), the primers used in the PCR are as follows:

ARO8-F：cattgttccttattcagttagCTAGCATGACTTTACCTGAATCAAAA(SEQ ID NO：4)；

ARO8-R：cgcatgatatctcgagctcagctagCCTATTTGGAAATACCAAATT(SEQ ID NO：5)。

in the step (3), the recombinant plasmid is a recombinant plasmid PYX212+ ARO8, and the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO: 3, respectively.

In order to solve the third technical problem, the invention discloses application of the saccharomyces cerevisiae engineering bacteria in fermentation for producing ethanol.

Wherein, the saccharomyces cerevisiae engineering bacteria can increase the biofilm yield in the process of producing ethanol by fermentation and is also within the protection scope of the invention.

Wherein, the saccharomyces cerevisiae engineering bacteria are activated and then fermented.

Wherein, the activation includes but is not limited to the activation method common in the prior art; preferably, the activation is to inoculate the saccharomyces cerevisiae engineering bacteria liquid into a YPD culture medium for culture and centrifugation; preferably, the activation is to inoculate the saccharomyces cerevisiae engineering bacteria liquid into YPD culture medium according to the volume ratio of 20-40%, culture for 18-30h at 20-40 ℃, and centrifuging; more preferably, the activation is to inoculate the saccharomyces cerevisiae engineering bacteria liquid with OD600 of 0.5-1.5 into YPD culture medium according to the volume ratio of 30 percent, culture for 24h at 30 ℃ and centrifuge; still more preferably, the activation is to inoculate the saccharomyces cerevisiae engineering bacteria liquid with OD600 of 1 in YPD culture medium according to the volume ratio of 30 percent, culture for 24h at 30 ℃ and centrifuge.

Wherein the formula of the fermentation medium comprises 60g/L glucose, 4g/L peptone, 4g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L yeast extract, 0.5g/L magnesium sulfate, 0.05g/L ferrous sulfate heptahydrate and 0.05g/L zinc sulfate heptahydrate.

Wherein the fermentation is any one of free fermentation, immobilized fermentation and embedded immobilized fermentation; preferably, the fermentation is immobilized fermentation or embedded immobilized fermentation; further preferably, the fermentation is an embedded immobilized fermentation.

Wherein the immobilized fermentation takes cotton fiber as an immobilized material; preferably, the dosage of the cotton fiber in the immobilized fermentation is 5-9g/100 mL; further preferably, the dosage of the cotton fiber in the immobilized fermentation is 7.5g/100 mL.

The embedding immobilization fermentation takes polyvinyl alcohol (PVA) as an embedding material, and adopts an adsorption method to co-immobilize glucoamylase and saccharomyces cerevisiae engineering bacteria.

Wherein, the embedding immobilization fermentation comprises the following steps:

(i) heating and boiling a mixture of polyvinyl alcohol, sodium alginate and water to obtain a first gel solution;

(ii) mixing the first gel solution, the saccharifying enzyme solution and the dry yeast aqueous solution of the saccharomyces cerevisiae engineering bacteria to obtain a second gel solution;

(iii) adding the second gel solution into the cross-linking agent solution to prepare immobilized gel particles;

(iv) and culturing and fermenting the obtained immobilized particles in a culture medium to obtain the ethanol.

In step (i), the polyvinyl alcohol has an average molecular weight of 20000-30000; preferably, the polyvinyl alcohol has an average molecular weight of 25000.

In the step (i), the concentration of the polyvinyl alcohol in the mixture is 0.005-0.015 g/mL; preferably, the concentration of polyvinyl alcohol in the mixture is 0.01 g/mL.

In the step (i), the concentration of sodium alginate in the mixture is 0.005-0.015 g/mL; preferably, the concentration of sodium alginate in the mixture is 0.01 g/mL.

In the step (ii), the enzyme activity of the saccharifying enzyme solution is 40000-60000U/g; preferably, the enzyme activity of the saccharifying enzyme solution is 50000U/g.

In the step (ii), the concentration of the dry yeast aqueous solution of the saccharomyces cerevisiae engineering bacteria is 5-15 g/L; preferably, the concentration of the dry yeast aqueous solution of the saccharomyces cerevisiae engineering bacteria is 10 g/L.

In the step (ii), the volume ratio of the first gel solution, the saccharifying enzyme solution and the dry yeast aqueous solution of the saccharomyces cerevisiae engineering bacteria is 1: 0.5-1.5: 0.5-1.5; preferably, the volume ratio of the first gel solution to the saccharifying enzyme solution to the dry yeast aqueous solution of the saccharomyces cerevisiae engineering bacteria is 1:1: 1.

in the step (iii), the cross-linking agent solution is a mixed solution of chitosan aqueous solution, acetic acid aqueous solution, boric acid and calcium chloride.

Wherein the concentration of the chitosan aqueous solution is 1% -3%; preferably, the concentration of the aqueous chitosan solution is 2%.

Wherein the viscosity of the chitosan aqueous solution is 0.5-1.5 Pa.s; preferably, the viscosity of the aqueous chitosan solution is 1 pa.s.

Wherein the concentration of the acetic acid aqueous solution is 1% -3%; preferably, the concentration of the acetic acid aqueous solution is 2%.

Wherein the volume ratio of the chitosan aqueous solution to the acetic acid aqueous solution is 1: 0.5-1.5; preferably, the volume ratio of the chitosan aqueous solution to the acetic acid aqueous solution is 1: 1.

in the step (iii), the concentration of calcium chloride in the cross-linking agent solution is 2% -5%; preferably, the concentration of calcium chloride in the crosslinker solution is 3.5%.

In the step (iii), the concentration of boric acid in the cross-linking agent solution is 1% -3%; preferably, the concentration of boric acid in the crosslinker solution is 2%.

In step (iv), the volume ratio of the immobilized particles to the fermentation medium is 1: 0.5-1.5; preferably, the volume ratio of the immobilized particles to the fermentation medium is 1: 1.

has the advantages that: compared with the prior art, the invention has the following advantages:

(1) according to the invention, the saccharomyces cerevisiae engineering bacteria for over-expressing the ARO8 saccharomyces cerevisiae gene are constructed, so that the cell permeability of the bacteria is improved, and the yield of the biofilm is improved, thereby improving the ethanol yield of the saccharomyces cerevisiae in the fermentation process, increasing the colony number and the adhesion, improving the sugar conversion rate and the ethanol yield, and shortening the fermentation period.

(2) According to the invention, the saccharifying enzyme preparation and the saccharomyces cerevisiae cells are co-immobilized by an embedding method, and on the basis of treatment of the permeability of cell membranes of the saccharomyces cerevisiae, the biomembrane yield and the glucose conversion rate of the saccharomyces cerevisiae ARO8 gene overexpression genetic engineering bacteria are further improved, the fermentation period is shortened, and the sugar consumption is reduced.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is an electrophoretogram of ARO8 gene.

FIG. 2 is a PCR electrophoresis image of ARO8 over-expressed colonies.

FIG. 3 shows the results of single batch immobilized fermentation of original strain S288c and modified strain S288c-ARO 8.

FIG. 4 shows the results of fermentation of S288c-ARO8 without pretreatment and after pretreatment.

FIG. 5 shows a single batch fermentation of immobilized S288c-ARO8 and embedded co-immobilized S288c-ARO 8.

FIG. 6 shows the crystal violet staining results of S288c and pre-treatment co-immobilized S288c-ARO 8.

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

In the following examples, the enzyme activities of the saccharifying enzyme solution and enzyme powder are defined as follows: 1mL of enzyme solution or 1g of enzyme powder is used as an enzyme activity unit for decomposing soluble starch to generate 1mg of glucose in 1 hour at the temperature of 40 ℃ and under the condition that the pH value is 4.6.

In the following examples, the percentages are by mass unless otherwise specified.

Example 1:

firstly, extracting the genome of the original Saccharomyces cerevisiae strain S288c

Using a fungal genome extraction kit (dr. gentle) of Takara corporation^TM(from Yeast)High Recovery)

(1) Taking an overnight culture solution of the Saccharomyces cerevisiae with OD1.2-1.8 into a system tube, centrifuging at 12000rpm for 1min, and removing a supernatant;

(2) adding 500 mu L of GenTLE Yeast Solution A into the precipitate, shaking easily, suspending the precipitate sufficiently at 37 ℃, and bathing for 1h, wherein the centrifugal tube is shaken gently for a plurality of times;

(3) adding 100 mu L of GenTLE Yeast Solution B, slightly shaking, uniformly mixing, and then carrying out water bath at 70 ℃ for 10 minutes;

(4) adding 200 mu L of GenTLE Yeast Solution C, slightly shaking, uniformly mixing, and standing on ice for 5 minutes;

(5) centrifuging the system tube at 4 ℃ and 12000rpm for 5 minutes;

(6) transferring the supernatant into a new system tube, adding 400 mu L of isopropanol, fully reversing and uniformly mixing;

(7) centrifuging the system tube at 4 ℃ and 12000rpm for 5 minutes, and removing supernatant;

(8) adding 500 μ L of precooled 70% ethanol into the precipitate, washing the precipitate upside down, and centrifuging at 12000rpm for 5 minutes at 4 ℃;

(9) discarding the supernatant, naturally drying the DNA at room temperature, and precipitating until no ethanol smell exists;

(10) adding a proper amount of TE Buffer/ddH₂And O. Genomic DNA was dissolved.

Secondly, amplifying a target gene ARO8 by PCR technology

(1) And (3) amplifying the ARO8 gene by adopting the Saccharomyces cerevisiae genome extracted in the first step as a template and adopting a PCR technology. The reaction system is shown in Table 1.

TABLE 1 PCR reaction System

The primers used in the PCR were as follows:

ARO8-F：cattgttccttattcagttagCTAGCATGACTTTACCTGAATCAAAA(SEQ ID NO：4)；

ARO8-R：cgcatgatatctcgagctcagctagCCTATTTGGAAATACCAAATT(SEQ ID NO：5)。

the PCR product (ARO8 gene fragment) was electrophoresed using 1.2% (1.2g/100mL) agarose gel, and the results are shown in FIG. 1.

(2) Gel recovery purification fragment ARO8 Takara kit (TaKaRa MiniBEST Agarose Gel DNA Extraction KitVer.4.0)

2.1 preparing agarose gel by using TBE buffer solution, and then carrying out agarose gel electrophoresis on the target fragment ARO 8;

2.2 cutting the agarose gel containing the target fragment ARO8 under an ultraviolet lamp, and completely absorbing the surface liquid by using a paper towel;

2.3 chopping the rubber blocks and weighing the rubber blocks. When the volume is calculated, the calculation is carried out according to 1mg to 1 μ L;

2.4 adding a dissolving solution Buffer GM into the rubber block, wherein the adding amount of the Buffer GM is less than that of the rubber block, uniformly mixing, dissolving the rubber block at the room temperature of 15-25 ℃, and at the moment, intermittently oscillating and mixing to fully dissolve the rubber block;

2.5 arranging Spin Colum in the kit on a collecting pipe;

2.6 transferring the solution obtained in the step 2.4 into Spin Column, centrifuging at 1200000 rpm for 1min, and discarding the filtrate;

2.7 adding 700 μ L Buffer WB into Spin Column, centrifuging at 12000rpm for 1min at room temperature, and discarding the filtrate;

2.8 repeating the operation step 2.7;

2.9 placing Spin Column on the collecting tube, centrifuging at 12000rpm for 1min at room temperature, and discarding the filtrate;

2.10 placing Spin Column in a new centrifuge tube of 1.5mL, adding 60 μ L of sterilized water at the center of the Spin Column membrane, and standing at room temperature for 1 min;

2.11 centrifugation at 12000rpm for 1min at room temperature to elute the ARO8 fragment;

2.12 carrying out agarose gel electrophoresis verification on the purified ARO8 target fragment, and calculating the concentration; wherein the nucleotide sequence of the ARO8 gene is shown in SEQ ID NO: 1.

thirdly, extracting the plasmid PYX212

Kit for upgrading plasmid using Axygen (AxyPrep)^TM Plasmid Miniprep Kit)

(1) A single colony of E.coli DH5 alpha (containing PYX212 and having a nucleotide sequence shown in SEQ ID NO: 2) was selected from a plate medium, wherein E.coli DH5 alpha was purchased from Shanghai Biotechnology engineering (Shanghai) GmbH, NO. B528413, and a method for storing PET-28a was performed according to the method in molecular cloning Experimental Manual (Huang Petang, et al, China, scientific Press, 2002, third edition); then inoculating into 5mL LB liquid culture medium containing antibiotics (resistance is 50 ug/mL kanamycin), and culturing at 37 ℃ overnight for 12-16 h;

(2) taking 2mL of overnight culture liquid, centrifuging at 12000rpm for 1min, and removing supernatant;

(3) the bacterial pellet was resuspended thoroughly with 250. mu.L of 4 ℃ precooled Buffer S1 (containing RNase A);

(4) adding 250 μ L Buffer S2, turning and mixing for 4-6 times to crack the thallus fully to form transparent solution;

(5) adding 350 mu L of Buffer S3, and slightly turning and mixing the mixture up and down for 6 to 8 times until compact coagulated masses are formed;

(6) centrifuging at 12000rpm for 10min at room temperature, and collecting supernatant;

(7) placing Spin Column in the kit on the Collection Tube;

(8) transferring the supernatant obtained in the step (6) into Spin Column, centrifuging at 12000rpm for 1min, and removing the filtrate;

(9) adding 500 μ L Buffer W1 into Spin Column, centrifuging at 12000rpm for 1min, and removing the filtrate;

(10) adding 700 mu L of Buffer W2 into Spin Column, centrifuging at 12000rpm for 1min, and removing the filtrate;

(11) repeating the operation step (10);

(12) placing Spin Column on Collection Tube again, centrifuging at 12000rpm for 1min, and removing residual lotion;

(13) placing Spin Column on new centrifuge tube of 1.5mL, adding 30-50 μ L of 65 deg.C sterilized water at center of Spin Column membrane, and standing at room temperature for 1 min;

(14) centrifuging at 12000rpm for 1min to elute DNA;

(15) the extracted plasmid PYX212 was verified by agarose gel electrophoresis, and the concentration was calculated.

Fourthly, the plasmid PYX212 is cut by using restriction enzyme NHE1

TABLE 2 restriction system

And (3) carrying out enzyme digestion at 37 ℃ for 30min, carrying out gel running verification, and carrying out gel recovery after confirming that the fragment is correct.

Fifthly, connecting the target gene ARO8 with the plasmid PYX212 after enzyme digestion to obtain a recombinant plasmid PYX212+ ARO8

Using Novozam one-step cloning kit (

II) the following reaction systems were prepared in an ice-water bath, see Table 3.

TABLE 3 enzyme Linked systems

After being bathed in water at 37 ℃ for 30min, the mixture is immediately ice-bathed for five minutes and stored at-20 ℃ for later use.

Sixthly, the recombinant plasmid PYX212+ ARO8 is transformed into E.coli DH5 alpha

(1) A tube of 200. mu.L E.coli DH 5. alpha. competent cell (No. B528413, Shanghai Biotechnology engineering, Ltd.) suspension was taken out of a refrigerator at-80 ℃ and thawed at room temperature, and immediately placed on ice after thawing;

(2) adding the solution of the recombinant plasmid PYX212+ ARO8 obtained in the step five into E.coli DH5 alpha competent cell suspension, shaking up gently, and placing on ice for 30 min;

(3) heating in 42 deg.C water bath for 45-90s, immediately cooling on ice for 5 min;

(4) adding 1mL LB liquid culture medium, mixing, and shake culturing at 37 deg.C for 40min to recover normal growth state of thallus;

(5) shaking the bacterial solution uniformly, coating 100 mu L of the bacterial solution on an LB resistance plate, inverting a culture dish, and culturing for 12 hours in a constant-temperature incubator at 37 ℃; the formula of the LB resistant plate is as follows: 10g/L of sodium chloride, 10g/L of tryptone, 5g/L of yeast powder and 20g/L of agar powder, sterilizing for 20min at 121 ℃, cooling and adding kanamycin to the final concentration of 50 mu g/mL;

(6) picking single colony from the resistant plate, preserving the bacteria, extracting the plasmid PYX212+ ARO8 according to the third step, carrying out agarose gel electrophoresis verification and sequencing verification on the extracted plasmid PET-PYX212+ ARO8, and obtaining a correct result, as shown in figure 2, wherein the nucleotide sequence is shown in SEQ ID NO: 3.

seventhly, the recombinant plasmid PYX212+ ARO8 is electrically transformed into the original saccharomyces cerevisiae

(1) Mixing 10 μ L PYX212+ ARO8 recombinant plasmid with Saccharomyces cerevisiae competence, and pre-cooling on ice for 30 min;

(2) transferring the mixed solution into a 2mm electric rotating cup, and carrying out ice bath for 5 minutes;

(3) wiping off moisture outside the electric rotating cup, putting the electric rotating cup into an electric rotating instrument, and carrying out electric rotation (voltage 1500V, capacitance 25 muF and resistance 200 omega);

(4) adding 1mL YPD culture medium into an ideal electric rotating cup, uniformly mixing, transferring into a 1.5mL centrifuge tube, and recovering for 1h at 30 ℃ and 200 rpm;

(5) and (4) centrifuging. Discarding 600 supernatant, using the rest 400 to resuspend and precipitate, taking 200 bacteria liquid to coat on a G418 resistant YPD plate, and culturing at 30 ℃ until bacterial colonies grow out;

(6) colony PCR verified whether the strain was a PYX212+ ARO8 recombinant Saccharomyces cerevisiae strain. The results are correct, see fig. 2, and the nucleotide sequence is shown in SEQ ID NO: 3.

example 2

(1) 100. mu.L of each of Glycerol strain S288c (outbreak) and S288c-ARO8 (over-expression strain constructed in example 1) was added to a sterilized 5mL of YPD liquid medium and cultured overnight for activation.

(2) According to the inoculation ratio of 10% in volume ratio, respectively embedding and immobilizing the activated S288c bacterial liquid and S288c-ARO8 bacterial liquid in the step (1), transferring to 100mL YPD liquid culture medium, continuously culturing at 30 ℃ at 200r/min until the OD600 of the bacterial liquid is between 0.8 and 1.2, centrifuging the obtained culture at 3000r/min for 10min, and then retaining the supernatant;

the specific operation method of embedding co-immobilization comprises the following steps: heating and boiling 0.2g of polyvinyl alcohol with the average molecular weight of 25000 and 0.2g of sodium alginate in 20mL of water to fully dissolve the polyvinyl alcohol and the sodium alginate to obtain a first gel solution, and then cooling the first gel solution to about 30 ℃; uniformly mixing the bacterial liquid to be treated, the diastase liquid (50000U/g) and the first gel solution according to the volume ratio in equal proportion to obtain a second gel solution; dropwise adding the obtained second gel solution into a cross-linking agent containing 3.5% of calcium chloride and 2% of boric acid aqueous solution, dropwise adding the second gel solution into the cross-linking agent to prepare immobilized gel particles with the diameter of about 3mm, filtering the immobilized gel particles after curing for a period of time, washing the immobilized gel particles with deionized water for three times, and immersing the immobilized gel particles into sterile water for later use to obtain an embedded immobilized microorganism carrier;

the preparation method of the cross-linking agent containing the 3.5% calcium chloride and 2% boric acid aqueous solution comprises the steps of taking a 2.0% chitosan aqueous solution with the viscosity of 1Pa.s, dissolving the chitosan aqueous solution in an isometric 2% acetic acid solution at 50 ℃ under a heat preservation condition, immediately adding boric acid and calcium chloride to enable the final concentrations of the boric acid and the calcium chloride to be 2% and 3.5% respectively, and dissolving the boric acid and the calcium chloride under a heat preservation condition to obtain the cross-linking agent.

(3) And (3) respectively taking 2mL of the two supernatants obtained in the step (2), measuring a light absorption value under OD600, and diluting the bacterial liquid by using a sterilized YPD liquid culture medium to enable the OD600 of the diluted bacterial liquid to be 0.01.

(4) Adding 200 mu L of the two bacterial liquids obtained in the step (3) into a 96-well plate, and culturing at 37 ℃ for 24 h.

(5) And pouring out the 96-well plate bacterial liquid, buffering for 3 times by using pure water, and patting dry.

(6) Adding 200 μ L of 1% crystal violet solution into 96-well plate, dyeing for 10min, washing with tap water, and air drying.

(7) After adding 200. mu.L of 33% glacial acetic acid into a 96-well plate for dissolution, gently shaking, measuring the biofilm yield by OD600, and taking an average value: the OD value of S288c in 96-well plate for 24 hr is 1.2-1.5, and the OD value of S288c-ARO8 over-expression strain in 96-well plate for 24 hr is 1.8-2.0. The change of the biofilm yield can be quantitatively observed through a crystal violet staining experiment, the experiment results are shown in FIG. 6, and the biofilm removal rate of Saccharomyces cerevisiae overexpressing ARO8 gene is obviously increased.

Example 3

(1) Each 100. mu.L of each of Glycerol bacteria S288c and S288c-ARO8 was added to 5mL of a sterilized YPD liquid medium for overnight culture and activation.

(2) Transferring the bacterial liquid obtained in the step (1) to 100mL YPD liquid culture medium according to the inoculation ratio of 10% by volume, and continuously culturing at 30 ℃ and 200r/min until the OD600 of the bacterial liquid is between 0.8 and 1.2.

(3) Respectively transferring the seed liquid obtained in the step (2) into 100mL of fermentation medium (the dosage of the cotton fiber material is 7.5g/100mL), carrying out immobilized fermentation at 35 ℃ and 200rpm/min, ending the reaction after the glucose is exhausted, measuring the residual sugar content of the fermentation liquid in each period by using a spectrophotometer, and measuring the content of alcohol in the fermentation liquid by using a high-efficiency gas chromatograph;

wherein the formula of the fermentation medium is as follows: 60g/L glucose, 4g/L peptone, 4g/L ammonium sulfate, 3g/L monopotassium phosphate, 3g/L yeast extract, 0.5g/L magnesium sulfate, 0.05g/L ferrous sulfate heptahydrate and 0.05g/L zinc sulfate heptahydrate.

Since the amount of biofilm is related to the production of ethanol and the consumption of glucose, the experiment qualitatively characterizes the change in production of biofilm by the production of ethanol and the consumption of glucose. The fermentation data show that the fermentation period of the original bacteria and the modified bacteria in the immobilized fermentation is obviously changed in comparison, wherein the immobilized fermentation result is shown in figure 3, the fermentation periods of the original bacteria S288c and the modified bacteria S288c-ARO8 are respectively 24h and 18h, and the fermentation period is shortened by about 6 h; and by analyzing the product ethanol yield data, the ethanol yields of the original strain S288c and the genetically modified strain S288c-ARO8 are about 83g/L and 94g/L at the fermentation end point, and the yield is improved by about 11g/L, so that the biofilm yield is improved.

Example 4: activation culture method

(1) The saccharomyces cerevisiae S288c-ARO8 is treated by an activation culture method, which is different from the activation method in the embodiment 3, and the specific operation steps are as follows:

an activation culture method: inoculating Saccharomyces cerevisiae strain S288c-ARO8 with OD600 of 1.0 into YPD culture medium at 30% by volume, culturing at 30 deg.C for 24h, centrifuging the obtained culture at 300r/min for 10min, and retaining supernatant to obtain strain liquid.

(2) Respectively transferring the bacterial liquid of the saccharomyces cerevisiae S288c-ARO8 treated by the activation culture method obtained in the step (1) and the bacterial liquid of the saccharomyces cerevisiae S288c-ARO8 which is not subjected to the activation culture method to 100mL of YPD liquid culture medium according to the inoculation ratio of 10% by volume, and continuously culturing at 30 ℃ and 200r/min until the OD600 of the bacterial liquid is between 0.8 and 1.2.

(3) Respectively transferring the seed liquid obtained in the step (2) into 100mL of fermentation culture medium, fermenting at 35 ℃ at 200rpm/min, ending the reaction after the glucose is exhausted, measuring the residual sugar content of the fermentation liquid in each period by using a spectrophotometer, and measuring the content of alcohol in the fermentation liquid by using a high-efficiency gas chromatograph.

The fermentation result is shown in fig. 4, and it can be seen that the fermentation period of the modified bacteria pretreated by the yeast activation culture method is not significantly changed compared with the modified bacteria not pretreated, but the ethanol yield is significantly improved, the final ethanol yield of the pretreated strain is about 80g/L, and is improved by 7g/L compared with 73g/L of the strain not pretreated.

Example 5: embedding co-immobilization fermentation

(1) Taking 2.0% chitosan water solution with viscosity of 1Pa.s, dissolving in 2% acetic acid solution of the same volume at 50 deg.C, immediately adding boric acid and calcium chloride to make the final concentrations of boric acid and calcium chloride respectively 2% and 3.5%, and dissolving at constant temperature to obtain the cross-linking agent solution.

(2) Weighing 120mg of saccharifying enzyme powder (50000U/g), adding 20mL of vinegar-sodium acetate buffer solution with pH of 4.5 for dissolving, repeatedly grinding insoluble substances by using a glass rod, pouring the dissolved liquid into a volumetric flask, washing residues by using buffer solution, repeating for 3 to 4 times, finally pouring all the liquid into the volumetric flask, fixing the volume by using the buffer solution, centrifuging by using a centrifugal machine at 4000rpm/min, and taking supernate, namely saccharifying enzyme liquid.

(3) Pretreating glycerol strain S288c and ARO8 modified strains (Saccharomyces cerevisiae S288c-ARO8) by the activation culture method in example 4, centrifuging, and removing supernatant to obtain active dry yeast; the obtained active dry yeast is prepared into 10/L active dry yeast aqueous solution for later use.

(4) 0.2g of polyvinyl alcohol having an average molecular weight of 25000 and 0.2g of sodium alginate were weighed, added to 20mL of deionized water, and heated to boil to obtain a first gel solution. And (3) preserving the temperature of the first gel solution at 34 ℃, adding the saccharifying enzyme solution (50000U/g, prepared in the step (2)) and the active dry yeast aqueous solution (prepared in the step (3)) with the concentration of 10g/L into the first gel solution, and uniformly mixing the saccharifying enzyme solution, the first gel solution and the yeast solution according to the ratio of 1:1:1 to obtain a second gel solution. And (2) sucking the second gel solution, dripping the second gel solution into a cross-linking agent solution (prepared in the step (1)) containing 3.5% of calcium chloride and 2% of boric acid to prepare immobilized gel particles with the diameter of about 3mm, solidifying for a period of time, filtering out the immobilized gel particles, washing with deionized water for three times, and immersing in sterile water for later use to obtain the immobilized yeast cell gel particles.

(5) The immobilized yeast cell gel particles are washed by sterile water for 6 times, and YPD culture medium with the same volume is added for proliferation culture. And (5) ventilating, shaking and culturing for 24h, and sealing for fermentation.

The fermentation result is shown in FIG. 5, compared with the over-expression strain of immobilized fermentation (example 3, fermentation period is 30h, ethanol yield is 94g/L), the fermentation time of embedding co-immobilization of the over-expression strain is 22h, 8h is shortened, the ethanol yield is 120g/L, and 26g/L is relatively improved; compared with 95g/L ethanol produced by embedding co-immobilization of original bacteria, the yield is improved by 25 g/L.

The invention provides a saccharomyces cerevisiae engineering bacterium for over-expressing an ARO8 gene, a construction method thereof, and ideas and methods for application thereof, and a plurality of methods and ways for specifically implementing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of industry

<120> saccharomyces cerevisiae engineering bacteria for over-expressing ARO8 gene and construction method and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1503

<212> DNA

<213> ARO8

<400> 1

atgactttac ctgaatcaaa agacttttct tacttgtttt cggatgaaac caatgctcgt 60

aaaccatccc cattgaaaac ctgcatccat cttttccaag atcctaacat tatctttttg 120

ggtggtggcc tgccattaaa agattatttc ccatgggata atctatctgt agattcaccc 180

aagcctcctt ttccccaggg tattggagct ccaattgacg agcagaattg cataaaatac 240

accgtcaaca aagattacgc tgataaaagt gccaatcctt ccaacgatat tcctttgtca 300

agagctttgc aatacgggtt cagtgctggt caacctgaac tattaaactt cattagagat 360

cataccaaga ttatccacga tttgaagtat aaggactggg acgttttagc cactgcaggt 420

aacacaaatg cctgggaatc tactttaaga gtcttttgta accgaggtga tgtcatctta 480

gttgaggcac attctttttc ctcttcattg gcttctgcag aggctcaagg tgtcattacc 540

ttccccgtgc caattgacgc tgatggtatc attcctgaaa aattagctaa agtcatggaa 600

aactggacac ctggtgctcc taaaccaaag ttgttataca ctattccaac gggccaaaat 660

ccaactggta cttccattgc agaccataga aaggaggcaa tttacaagat cgctcaaaag 720

tacgacttcc taattgtgga agatgaacct tattatttct tacaaatgaa tccctacatc 780

aaagacttga aggaaagaga gaaggcacaa agttctccaa agcaggacca tgacgaattt 840

ttgaagtcct tggcaaacac tttcctttcc ttggatacag aaggccgtgt tattagaatg 900

gattcctttt caaaagtttt ggccccaggg acaagattgg gttggattac tggttcatcc 960

aaaatcttga agccttactt gagtttgcat gaaatgacga ttcaagcccc agcaggtttt 1020

acacaagttt tggtcaacgc tacgctatcc aggtggggtc aaaagggtta cttggactgg 1080

ttgcttggcc tgcgtcatga atacactttg aaacgtgact gtgccatcga tgccctttac 1140

aagtatctac cacaatctga tgctttcgtg atcaatcctc caattgcagg tatgtttttc 1200

accgtgaaca ttgacgcatc tgtccaccct gagtttaaaa caaaatacaa ctcagaccct 1260

taccagctag aacagagtct ttaccacaaa gtggttgaac gtggtgtttt agtggttccc 1320

ggttcttggt tcaagagtga gggtgagacg gaacctcctc aacccgctga atctaaagaa 1380

gtcagtaatc caaacataat tttcttcaga ggtacctatg cagctgtctc tcctgagaaa 1440

ctgactgaag gtctgaagag attaggtgat actttatacg aagaatttgg tatttccaaa 1500

tag 1503

<210> 2

<211> 9996

<212> DNA

<213> plasmid (PYX212)

<400> 2

ctgtatgtgt tttttgtagt tatagattta agcaagaaaa gaatacaaac aaaaaattga 60

aaaagattga tttagaatta aaaagaaaaa tatttacgta agaagggaaa atagtaaatg 120

ttgcaagttc actaaactcc taaattatgc tgccctttat attccctgtt acagcagccg 180

agccaaaggt atataggctc ctttgcatta gcatgcgtaa caaaccacct gtcagtttca 240

accgaggtgg tatccgagag aattgtgtga ttgctttaat taatttcgga gaatctcaca 300

tgccactgaa gattaaaaac tggatgccag aaaaggggtg tccgagtgta acatcaatag 360

aggaagctga aaagtcttag aacgggtaat cttccaccaa cctgatgggt tcctagatat 420

aatctcgaag ggaataagta gggtgatacc gtcagaagtg tctgaatgta ttgaggtcct 480

cacagtttaa atcccgctca cactaacgta ggattattat aactcaaaaa aatggcatta 540

ttctaagtaa gttaaatatc cgtaatcttt aaacactatg tagttaggtc tccctcacaa 600

tcagtccatt tgggtagcac ggtcctcagg acgtatctat tgatggattc gtccagttcc 660

atcaccatta cgctcccgtt aggaacattg gtaaacgatt caaactcttc gtatgtccat 720

ctaaaccatt tcatcaggaa tactctggaa taaataccat gtgtaactag gacaacaaca 780

tctctgggtc ttctctcttg cctatcatgg aagtgcctga ataaagtctc ttggaaactg 840

gcgactctgt catatacatc tgccgcactt ctccatgagg gaatctgaag aagaatgacc 900

atacgtagat ccccaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg 960

ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 1020

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatcgc tcatgagaca 1080

ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 1140

ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 1200

aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 1260

actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 1320

gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 1380

agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt acaccagtca 1440

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagttcc tgccataacc 1500

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaagagctaa 1560

ccgctttttt tcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 1620

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gccatcaagc tcgacggccg 1680

gcccggtacc ataacttcgt atagcataca ttatacgaag ttatttgaag tcggacagtg 1740

agtgtagtct tgagaaattc tgaagccgta tttttattat cagtgagtca gtcatcagga 1800

gatcctctac gccggacgca tcgtggccga cctgcagggg gggggggggc gctgaggtct 1860

gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca 1920

gaaagtgagg gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt 1980

gaacttttgc tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt 2040

caactcagca aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg 2100

ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa 2160

tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc 2220

tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg 2280

tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata 2340

aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc 2400

ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca 2460

ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga 2520

tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc 2580

agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt 2640

ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 2700

atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 2760

tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 2820

tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 2880

tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga 2940

atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat 3000

gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt 3060

tccccccccc ccctgcaggt cggcatcacc ggcgccacag gtgcggttgc tggcgcctat 3120

atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt 3180

ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg 3240

catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggcataact 3300

tcgtatagca tacattatac gaagttatgg tacccaattc gccctatagt gagtcgtatt 3360

tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 3420

attccacaca acataggagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 3480

aggtaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 3540

tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 3600

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgaccggta 3660

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 3720

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3780

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3840

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 3900

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 3960

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4020

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4080

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4140

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4200

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4260

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4320

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4380

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4440

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 4500

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 4560

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 4620

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 4680

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 4740

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 4800

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 4860

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 4920

tcaaggcgag ttacatgatc ccccatgttg tccaaaaaag cggttagctc cttcggtcct 4980

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5040

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5100

accaagtcat tctgagaata gtgtatgcgg cgagcgagtt gctcttgccc ggcgtcaata 5160

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5220

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 5280

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 5340

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 5400

atactcttcc tttttcaata ttattgaagc atttatcacg gttattgtct catgagcgga 5460

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 5520

aaagtgccac ctgacgtact agtgagagtg cggccgcagc tttgaagaaa aatgcgcctt 5580

attcaatctt tgctataaaa aatggcccaa aatctcacat tggaagacat ttgatgacct 5640

catttctttc aatgaagggc ctaacggagt tgactaatgt tgtgggaaat tggagcgata 5700

agggtgcttc tgccgtggcc aggacaacgt atactcatca gataacagca atacctgatc 5760

actacttcgc actagtttct cggtactatg catatgatcc aatatcaaag gaaatgatag 5820

cattgaagga tgagactaat ccaattgagg agtggcagca tatagaacag ctaaagggta 5880

gtgctgaagg aagcatacga taccccgcat ggaatgggat aatatcacag gaggtactag 5940

actacctttc atcctacata aatagacgca tataagtacg catttaagca taaacacgca 6000

ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata ggtgcgacgt 6060

gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa 6120

acgctttgaa gttcctattc cgaagttcct attctctaga aagtatagga acttcagagc 6180

gcttttgaaa accaaaagcg ctctgaagac gcactttcaa aaaaccaaaa acgcaccgga 6240

ctgtaacgag ctactaaaat attgcgaata ccgcttccac aaacattgct gaaaagtatc 6300

tctttgctat atatctctgt gctatatccc tatataacct acccatccac ctttcgctcc 6360

ttgaacttgc atctaaactc gacctctaca ttttttatgt ttatctctag tattactctt 6420

tagacaaaaa aattgtagta agaactattc atagagtgaa tcgaaaacaa tacgaaaatg 6480

taaacatttc ctatacgtag tatatagaga caaaatagaa gaaaccgttc ataattttct 6540

gaccaatgaa gaatcatcaa cgctatcact ttctgttcac aaagtatgcg gaatccacat 6600

cggtatagaa tataatcggg gatgccttta tcttgaaaaa atgcacccgc agcttcgcta 6660

gtaatcagta aacgcgggaa gtggagtcag gcttttttta tggaagagaa aatagacacc 6720

aaagtagcct tcttctaacc ttaacggacc tacagtgcaa aaagttatca agagactgca 6780

ttatagagcg cacaaaggag aaaaaaagta atctaagatg ctttgttaga aaaatagcgc 6840

tctcgggatg catttttgta gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc 6900

gctctcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 6960

agcgctctcg cgttgcattt ttgttttaca aaaatgaagc acagattctt cgttggtaaa 7020

atagcgcttt cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa 7080

aaattagcgc tctcgcgttg catttttgtt ctacaaaatg aagcacagat gcttcgttaa 7140

caaagatatg ctattgaagt gcaagatgga aacgcagaaa atgaaccggg gatgcgacgt 7200

gcaagattac ctatgcaata gatgcaatag tttctccagg aaccgaaata catacattgt 7260

cttccgtaaa gcgctagact atatattatt atacaggttc aaatatacta tctgtttcag 7320

ggaaaactcc caggttcgga tgttcaaaat tcaatgatgg gtaacaagta cgatcgtaaa 7380

tctgtaaaac agtttgtcgg atattaggct gtatctcctc aaagcgtatt cgaatatcat 7440

tgagaagctg cagcgtcaca tcggataata atgatggcag ccattgtaga agtgcctttt 7500

gcatttctag tctctttctc ggtctagcta gttttactac atcgcgaaga tagaatctta 7560

gatcacactg cctttgctga gctggatcaa tagagtaaca aaagagtggt aaggcctcgt 7620

taaaggacaa ggacctgagc ggaagtgtat cgtacagtag acggagtata ctagtatagt 7680

ctatagtccg tggaattgat ccactagttc tagagcggcc gcacgtctaa gaaaccatta 7740

ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 7800

tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 7860

tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 7920

gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatacca 7980

cagcttttca attcaattca tcattttttt tttattcttt tttttgattt cggtttcttt 8040

gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag gagcacagac 8100

ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc ccagtattct 8160

taacccaact gcacagaaca aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta 8220

catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca 8280

tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac 8340

tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatat 8400

tgactgattt ttcgatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca 8460

attttttact cttcgaagac agaaaatttg ctgacattgg taatacagtc aaattgcagt 8520

actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgc 8580

tggggccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta 8640

gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata 8700

ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc 8760

aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg 8820

gtttagatga caagggagac gcattgggtc aacagtatag aaccgtggat gatgtggtct 8880

ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta 8940

aggtacaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc 9000

agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag 9060

cttcaattta attatatcag ttattaccct atgcggtgtg aaataccgca cagatgcgta 9120

aggagaaaat accgcatcag gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa 9180

atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 9240

aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 9300

tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 9360

cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaaccactaa 9420

atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 9480

cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 9540

tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc 9600

gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc 9660

tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag 9720

ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgaattaa ccctcactaa 9780

agggaacaaa agctggagct agacaaagac aaaaaaaaaa agggatgtat cggtcagtca 9840

ttaatactag gaacaagaga aaggaaaaac gttcattgtt ccttattcag ttagctagcg 9900

ctagctgagc tcgagatatc atgcgtagtc aggcacatca tacggatacc cgggtcgacg 9960

cgtaagcttg tgggccctag gatccatggt gaattc 9996

<210> 3

<211> 11499

<212> DNA

<213> recombinant plasmid (PYX212+ ARO8)

<400> 3

ctgtatgtgt tttttgtagt tatagattta agcaagaaaa gaatacaaac aaaaaattga 60

aaaagattga tttagaatta aaaagaaaaa tatttacgta agaagggaaa atagtaaatg 120

ttgcaagttc actaaactcc taaattatgc tgccctttat attccctgtt acagcagccg 180

agccaaaggt atataggctc ctttgcatta gcatgcgtaa caaaccacct gtcagtttca 240

accgaggtgg tatccgagag aattgtgtga ttgctttaat taatttcgga gaatctcaca 300

tgccactgaa gattaaaaac tggatgccag aaaaggggtg tccgagtgta acatcaatag 360

aggaagctga aaagtcttag aacgggtaat cttccaccaa cctgatgggt tcctagatat 420

aatctcgaag ggaataagta gggtgatacc gtcagaagtg tctgaatgta ttgaggtcct 480

cacagtttaa atcccgctca cactaacgta ggattattat aactcaaaaa aatggcatta 540

ttctaagtaa gttaaatatc cgtaatcttt aaacactatg tagttaggtc tccctcacaa 600

tcagtccatt tgggtagcac ggtcctcagg acgtatctat tgatggattc gtccagttcc 660

atcaccatta cgctcccgtt aggaacattg gtaaacgatt caaactcttc gtatgtccat 720

ctaaaccatt tcatcaggaa tactctggaa taaataccat gtgtaactag gacaacaaca 780

tctctgggtc ttctctcttg cctatcatgg aagtgcctga ataaagtctc ttggaaactg 840

gcgactctgt catatacatc tgccgcactt ctccatgagg gaatctgaag aagaatgacc 900

atacgtagat ccccaattct tgaagacgaa agggcctcgt gatacgccta tttttatagg 960

ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 1020

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatcgc tcatgagaca 1080

ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 1140

ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 1200

aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 1260

actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 1320

gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 1380

agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt acaccagtca 1440

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagttcc tgccataacc 1500

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaagagctaa 1560

ccgctttttt tcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 1620

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gccatcaagc tcgacggccg 1680

gcccggtacc ataacttcgt atagcataca ttatacgaag ttatttgaag tcggacagtg 1740

agtgtagtct tgagaaattc tgaagccgta tttttattat cagtgagtca gtcatcagga 1800

gatcctctac gccggacgca tcgtggccga cctgcagggg gggggggggc gctgaggtct 1860

gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca 1920

gaaagtgagg gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt 1980

gaacttttgc tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt 2040

caactcagca aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg 2100

ctctgccagt gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa 2160

tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc 2220

tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg 2280

tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata 2340

aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc 2400

ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca 2460

ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga 2520

tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc 2580

agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt 2640

ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg 2700

atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca 2760

tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca 2820

tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca 2880

tataaatcag catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga 2940

atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat 3000

gatgatatat ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt 3060

tccccccccc ccctgcaggt cggcatcacc ggcgccacag gtgcggttgc tggcgcctat 3120

atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt 3180

ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg 3240

catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggcataact 3300

tcgtatagca tacattatac gaagttatgg tacccaattc gccctatagt gagtcgtatt 3360

tcgagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 3420

attccacaca acataggagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 3480

aggtaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 3540

tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 3600

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgaccggta 3660

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 3720

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3780

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3840

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 3900

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 3960

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4020

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4080

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4140

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4200

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4260

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4320

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4380

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4440

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 4500

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 4560

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 4620

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 4680

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 4740

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 4800

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 4860

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 4920

tcaaggcgag ttacatgatc ccccatgttg tccaaaaaag cggttagctc cttcggtcct 4980

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5040

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5100

accaagtcat tctgagaata gtgtatgcgg cgagcgagtt gctcttgccc ggcgtcaata 5160

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5220

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 5280

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 5340

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 5400

atactcttcc tttttcaata ttattgaagc atttatcacg gttattgtct catgagcgga 5460

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 5520

aaagtgccac ctgacgtact agtgagagtg cggccgcagc tttgaagaaa aatgcgcctt 5580

attcaatctt tgctataaaa aatggcccaa aatctcacat tggaagacat ttgatgacct 5640

catttctttc aatgaagggc ctaacggagt tgactaatgt tgtgggaaat tggagcgata 5700

agggtgcttc tgccgtggcc aggacaacgt atactcatca gataacagca atacctgatc 5760

actacttcgc actagtttct cggtactatg catatgatcc aatatcaaag gaaatgatag 5820

cattgaagga tgagactaat ccaattgagg agtggcagca tatagaacag ctaaagggta 5880

gtgctgaagg aagcatacga taccccgcat ggaatgggat aatatcacag gaggtactag 5940

actacctttc atcctacata aatagacgca tataagtacg catttaagca taaacacgca 6000

ctatgccgtt cttctcatgt atatatatat acaggcaaca cgcagatata ggtgcgacgt 6060

gaacagtgag ctgtatgtgc gcagctcgcg ttgcattttc ggaagcgctc gttttcggaa 6120

acgctttgaa gttcctattc cgaagttcct attctctaga aagtatagga acttcagagc 6180

gcttttgaaa accaaaagcg ctctgaagac gcactttcaa aaaaccaaaa acgcaccgga 6240

ctgtaacgag ctactaaaat attgcgaata ccgcttccac aaacattgct gaaaagtatc 6300

tctttgctat atatctctgt gctatatccc tatataacct acccatccac ctttcgctcc 6360

ttgaacttgc atctaaactc gacctctaca ttttttatgt ttatctctag tattactctt 6420

tagacaaaaa aattgtagta agaactattc atagagtgaa tcgaaaacaa tacgaaaatg 6480

taaacatttc ctatacgtag tatatagaga caaaatagaa gaaaccgttc ataattttct 6540

gaccaatgaa gaatcatcaa cgctatcact ttctgttcac aaagtatgcg gaatccacat 6600

cggtatagaa tataatcggg gatgccttta tcttgaaaaa atgcacccgc agcttcgcta 6660

gtaatcagta aacgcgggaa gtggagtcag gcttttttta tggaagagaa aatagacacc 6720

aaagtagcct tcttctaacc ttaacggacc tacagtgcaa aaagttatca agagactgca 6780

ttatagagcg cacaaaggag aaaaaaagta atctaagatg ctttgttaga aaaatagcgc 6840

tctcgggatg catttttgta gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc 6900

gctctcgcgt tgcatttctg ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt 6960

agcgctctcg cgttgcattt ttgttttaca aaaatgaagc acagattctt cgttggtaaa 7020

atagcgcttt cgcgttgcat ttctgttctg taaaaatgca gctcagattc tttgtttgaa 7080

aaattagcgc tctcgcgttg catttttgtt ctacaaaatg aagcacagat gcttcgttaa 7140

caaagatatg ctattgaagt gcaagatgga aacgcagaaa atgaaccggg gatgcgacgt 7200

gcaagattac ctatgcaata gatgcaatag tttctccagg aaccgaaata catacattgt 7260

cttccgtaaa gcgctagact atatattatt atacaggttc aaatatacta tctgtttcag 7320

ggaaaactcc caggttcgga tgttcaaaat tcaatgatgg gtaacaagta cgatcgtaaa 7380

tctgtaaaac agtttgtcgg atattaggct gtatctcctc aaagcgtatt cgaatatcat 7440

tgagaagctg cagcgtcaca tcggataata atgatggcag ccattgtaga agtgcctttt 7500

gcatttctag tctctttctc ggtctagcta gttttactac atcgcgaaga tagaatctta 7560

gatcacactg cctttgctga gctggatcaa tagagtaaca aaagagtggt aaggcctcgt 7620

taaaggacaa ggacctgagc ggaagtgtat cgtacagtag acggagtata ctagtatagt 7680

ctatagtccg tggaattgat ccactagttc tagagcggcc gcacgtctaa gaaaccatta 7740

ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 7800

tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 7860

tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 7920

gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatacca 7980

cagcttttca attcaattca tcattttttt tttattcttt tttttgattt cggtttcttt 8040

gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag gagcacagac 8100

ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc ccagtattct 8160

taacccaact gcacagaaca aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta 8220

catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca 8280

tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac 8340

tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatat 8400

tgactgattt ttcgatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca 8460

attttttact cttcgaagac agaaaatttg ctgacattgg taatacagtc aaattgcagt 8520

actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgc 8580

tggggccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta 8640

gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata 8700

ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc 8760

aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg 8820

gtttagatga caagggagac gcattgggtc aacagtatag aaccgtggat gatgtggtct 8880

ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta 8940

aggtacaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc 9000

agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag 9060

cttcaattta attatatcag ttattaccct atgcggtgtg aaataccgca cagatgcgta 9120

aggagaaaat accgcatcag gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa 9180

atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 9240

aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 9300

tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 9360

cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaaccactaa 9420

atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 9480

cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 9540

tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcgc 9600

gccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc 9660

tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag 9720

ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgaattaa ccctcactaa 9780

agggaacaaa agctggagct agacaaagac aaaaaaaaaa agggatgtat cggtcagtca 9840

ttaatactag gaacaagaga aaggaaaaac gttcattgtt ccttattcag ttagctagca 9900

tgactttacc tgaatcaaaa gacttttctt acttgttttc ggatgaaacc aatgctcgta 9960

aaccatcccc attgaaaacc tgcatccatc ttttccaaga tcctaacatt atctttttgg 10020

gtggtggcct gccattaaaa gattatttcc catgggataa tctatctgta gattcaccca 10080

agcctccttt tccccagggt attggagctc caattgacga gcagaattgc ataaaataca 10140

ccgtcaacaa agattacgct gataaaagtg ccaatccttc caacgatatt cctttgtcaa 10200

gagctttgca atacgggttc agtgctggtc aacctgaact attaaacttc attagagatc 10260

ataccaagat tatccacgat ttgaagtata aggactggga cgttttagcc actgcaggta 10320

acacaaatgc ctgggaatct actttaagag tcttttgtaa ccgaggtgat gtcatcttag 10380

ttgaggcaca ttctttttcc tcttcattgg cttctgcaga ggctcaaggt gtcattacct 10440

tccccgtgcc aattgacgct gatggtatca ttcctgaaaa attagctaaa gtcatggaaa 10500

actggacacc tggtgctcct aaaccaaagt tgttatacac tattccaacg ggccaaaatc 10560

caactggtac ttccattgca gaccatagaa aggaggcaat ttacaagatc gctcaaaagt 10620

acgacttcct aattgtggaa gatgaacctt attatttctt acaaatgaat ccctacatca 10680

aagacttgaa ggaaagagag aaggcacaaa gttctccaaa gcaggaccat gacgaatttt 10740

tgaagtcctt ggcaaacact ttcctttcct tggatacaga aggccgtgtt attagaatgg 10800

attccttttc aaaagttttg gccccaggga caagattggg ttggattact ggttcatcca 10860

aaatcttgaa gccttacttg agtttgcatg aaatgacgat tcaagcccca gcaggtttta 10920

cacaagtttt ggtcaacgct acgctatcca ggtggggtca aaagggttac ttggactggt 10980

tgcttggcct gcgtcatgaa tacactttga aacgtgactg tgccatcgat gccctttaca 11040

agtatctacc acaatctgat gctttcgtga tcaatcctcc aattgcaggt atgtttttca 11100

ccgtgaacat tgacgcatct gtccaccctg agtttaaaac aaaatacaac tcagaccctt 11160

accagctaga acagagtctt taccacaaag tggttgaacg tggtgtttta gtggttcccg 11220

gttcttggtt caagagtgag ggtgagacgg aacctcctca acccgctgaa tctaaagaag 11280

tcagtaatcc aaacataatt ttcttcagag gtacctatgc agctgtctct cctgagaaac 11340

tgactgaagg tctgaagaga ttaggtgata ctttatacga agaatttggt atttccaaat 11400

aggctagctg agctcgagat atcatgcgta gtcaggcaca tcatacggat acccgggtcg 11460

acgcgtaagc ttgtgggccc taggatccat ggtgaattc 11499

<210> 4

<211> 47

<212> DNA

<213> primer (ARO8-F)

<400> 4

cattgttcct tattcagtta gctagcatga ctttacctga atcaaaa 47

<210> 5

<211> 46

<212> DNA

<213> primer (ARO8-R)

<400> 5

cgcatgatat ctcgagctca gctagcctat ttggaaatac caaatt 46

Claims

1. An engineering bacterium of saccharomyces cerevisiae is characterized in that a recombinant plasmid carrying an ARO8 gene is introduced into original saccharomyces cerevisiae.

2. The engineered Saccharomyces cerevisiae strain of claim 1, wherein the original Saccharomyces cerevisiae is Saccharomyces cerevisiae S288 c.

3. The engineered saccharomyces cerevisiae strain of claim 1, wherein the recombinant plasmid carrying ARO8 gene is a PYX212 recombinant plasmid carrying ARO8 gene.

4. The construction method of the saccharomyces cerevisiae engineering bacteria of any one of the claims 1 to 3, characterized by comprising the following steps:

(1) amplifying an ARO8 gene by taking the genome of the original saccharomyces cerevisiae as a template;

(2) inserting the gene obtained in the step (1) into a PYX212 plasmid to obtain a PYX212 recombinant plasmid carrying an ARO8 gene;

5. Use of the engineered saccharomyces cerevisiae strain of any one of claims 1-3 in the production of ethanol by fermentation.

6. Use according to claim 5, wherein biofilm production is increased in a fermentative ethanol production process.

7. The use of claim 6, wherein the fermentation medium is formulated with 60g/L glucose, 4g/L peptone, 4g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 3g/L yeast extract, 0.5g/L magnesium sulfate, 0.05g/L ferrous sulfate heptahydrate, 0.05g/L zinc sulfate heptahydrate.

8. Use according to claim 6, wherein the fermentation is an immobilized fermentation.

9. The use according to claim 6, wherein the fermentation is an embedded immobilized fermentation.

10. The application of claim 9, wherein polyvinyl alcohol is used as an embedding material, and the saccharifying enzyme and the saccharomyces cerevisiae engineering bacteria are co-immobilized by an adsorption method.