CN110904123A - Gene for promoting saccharomyces cerevisiae to produce cane molasses alcohol with high yield and application of gene - Google Patents
Gene for promoting saccharomyces cerevisiae to produce cane molasses alcohol with high yield and application of gene Download PDFInfo
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- CN110904123A CN110904123A CN201910405322.7A CN201910405322A CN110904123A CN 110904123 A CN110904123 A CN 110904123A CN 201910405322 A CN201910405322 A CN 201910405322A CN 110904123 A CN110904123 A CN 110904123A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
- C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
- C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention belongs to the technical field of biology, and relates to a gene PHO4 for promoting saccharomyces cerevisiae to produce cane molasses alcohol with high yield, and a recombinant high-yield strain of the gene PHO4 for promoting saccharomyces cerevisiae to produce high-concentration alcohol by taking cane molasses as a raw material. The strain is preserved in China center for type culture collection (CCTCC M2018917), the preservation date is 12 months and 21 days in 2018, the taxonomic name is Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the strain number is MF01-PH 04. The gene PHO4 of the present invention has no report on the use of the gene in this respect. The 56-hour yield of cane molasses alcohol of the invention reaches the highest value of 114.71 g per liter, the fermentation period is short, the alcohol content is high, and the strain is suitable for high-concentration alcohol fermentation of cane molasses, thereby improving the utilization rate of raw materials, reducing the production cost of alcohol, and bringing remarkable economic benefits to the alcohol fermentation industry.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a gene for promoting saccharomyces cerevisiae to perform high-concentration alcohol fermentation by utilizing cane molasses, a construction method of a recombinant high-yield yeast strain thereof and application of the gene in producing fuel alcohol.
Background
Biofuel alcohol is by far the most successful liquid alternative fuel as a renewable energy source. How to develop and utilize fuel alcohol is the focus of common attention of countries in the world at present. China is no exception, and non-grain biofuel alcohol is used as a strategic emerging industry. Cane molasses is a byproduct of sucrose production and has become a main raw material for producing fuel alcohol in southern areas of China, such as Guangxi province. In fact, the sugar cane molasses fuel alcohol industry has become an emerging dominant industry in Guangxi agriculture. At present, the sugar cane molasses fuel alcohol industry still commonly has two prominent problems of low alcohol fermentation level, serious environmental pollution and the like, and the most important is lack of high-performance industrial strains. Aiming at the two problems of sugar molasses alcohol in China and combining the actual situation of Guangxi, the invention discloses a series of wild type saccharomyces cerevisiae industrial strains with different alcohol fermentation performances/phenotypes (alcohol concentration, fermentation speed, strain growth rate, sugar utilization capacity, respiratory strength and the like) which are selected from wastes of a Guangxi sugar cane sugar factory in the early period: the yeast flora with low yield, medium yield, high yield and the like establishes a relatively complete strain bank aiming at the fermentation of cane molasses alcohol. The highly productive strain had the following phenotype: the alcohol content is highest, but the growth is slowest (the fermentation period is long), while the alcohol content of the low-yield strain is lowest, but the low-yield strain has the advantages of fastest growth and fastest sugar utilization (the fermentation period is short), and the performance of the medium-yield strain is between the two. The high-yield strain is applied to annual production of 5 ten thousand tons of cane molasses alcohol, molasses alcohol fermentation can be carried out at high temperature (37 ℃) in summer, the alcohol content reaches 94g/L, energy consumption is saved, and production benefits are remarkable.
In the early period of the team, the comprehensive comparative analysis of the fermentation performance (phenotype) of each strain of the high-yield, medium-yield and low-yield 3 series yeast floras is carried out, 1 strain of each high-yield, medium-yield and low-yield yeast is selected from the comprehensive comparative analysis to carry out whole genome re-sequencing and comparative genomics analysis, the high-yield yeast is found to have stronger capacity of accepting heterologous genes, and 56 key regulatory genes which are possibly identical to the high-yield cane molasses ethanol are excavated. The PH04 gene belongs to one of the 56 genes, is the most important positive control gene in the phosphate control system (PHOregulon), and is located on the sixth chromosome (VI). The invention introduces PH04 gene of low-yield strain MC15 into high-yield wild strain MF01, replaces PH04 gene of original high-yield strain MF01 by gene homologous recombination,
obtains the PH04 gene recombinant high-yield strain which can rapidly utilize cane molasses for high-concentration alcohol fermentation compared with the original high-yield wild strain MF 01. The invention can realize high-concentration alcohol fermentation, shorten the alcohol fermentation time, save the energy consumption and has great significance for molasses alcohol production.
Disclosure of Invention
The invention provides a gene for promoting saccharomyces cerevisiae to ferment high-concentration alcohol by utilizing cane molasses and a construction method of a recombinant high-yield yeast strain thereof. The recombinant high-yield strain can be well applied to the fermentation production of cane molasses alcohol.
The technical scheme of the invention is as follows: the gene for promoting saccharomyces cerevisiae to perform high-concentration alcohol fermentation by utilizing cane molasses is PH 04.
The strain capable of quickly utilizing the cane molasses as the raw material to perform high-concentration alcohol fermentation is MF01-PH04, is named as Saccharomyces cerevisiae (Saccharomyces cerevisiae), is preserved in a China center for type culture collection in 12 months and 21 days in 2018, has a preservation number of CCTCC M2018917, and is preserved in Wuhan city Hongshan eight-path Wuhan university in Wuhan mountainous area.
The construction method of the saccharomyces cerevisiae strain capable of rapidly utilizing the cane molasses as the raw material for high-concentration alcohol fermentation comprises the following steps: the PH04 gene of the low-yield yeast MC15 is cloned by PCR, and is electrically transformed and introduced into a haploid protoplast of a high-yield yeast MF01 to obtain a recombinant high-yield yeast MF01-PH04, and the PH04 gene is amplified after passage to 30 generations and is found to be stable by sequencing.
The application of the saccharomyces cerevisiae strain MF01-PH04 in the fermentation production of high-concentration alcohol of cane molasses comprises the following steps: 5 percent of inoculation amount is added into saccharomyces cerevisiae in 20-degree Bx molasses, after the saccharomyces cerevisiae is cultured at 30 ℃ and 180rpm to form seed liquid, high-concentration molasses with the same volume of 50-degree Bx is added for culturing for 24 hours, standing and fermenting are carried out, the yield of alcohol reaches the maximum value of 114.71 grams per liter after 32 hours of fermentation, the fermentation time is shortened by 8 hours compared with the original high-yield strain, and the alcohol content is improved by 5.3 percent.
The invention has the outstanding advantages that: the gene capable of rapidly growing and utilizing cane molasses as raw material to perform high-concentration alcohol fermentation is PH04, and a phosphorus system positive regulation gene, and the application of the gene in the aspect is not reported at present; compared with the original high-yield yeast strain MF01, the obtained Saccharomyces cerevisiae MF01-PH04 has the advantages that the fermentation time is shortened by 8 hours compared with the original high-yield strain, the alcohol content is improved by 5.3%, the alcohol yield reaches the highest value of 114.71 g/L, the fermentation period is shorter than that of common industrial yeasts, the alcohol content is high, the method is suitable for high-concentration alcohol fermentation of cane molasses, the utilization rate of raw materials is improved, the alcohol production cost is reduced, and remarkable economic benefits can be brought to the alcohol fermentation industry.
Drawings
FIG. 1 shows agarose gel electrophoresis of PCR amplification product of PH04 gene of low-yield yeast MC15 (ML: DL2000 DNAmarker; 1, 2: PH04 gene; M: 1kb DNA Ladder Marker) with gene size of 1139 bp.
FIG. 2 shows the difference between the gas production of recombinant high-yielding yeast MF01-PH04 and the gas production of the original high-yielding strain MF01, 2 in parallel.
FIG. 3 is the fermentation curve of sugar cane molasses alcohol of recombinant high-yield yeast MF01-PH04, original high-yield strain MF01 and low-yield strain MC 15.
Detailed Description
Example 1: the PH04 gene of the low-yield yeast MC15 was cloned.
1. And (3) extracting the genomic DNA of the low-yield yeast MC 15.
Medium YPD: 2% glucose, 2% peptone and 1% yeast powder.
Activating and culturing yeast overnight, collecting 2mL thallus precipitate, washing with water for 2 times, adding 250 microliters of lysate, 100 microliters of 1XTE, and sterile quartz sand with about one third of the volume of the liquid, shaking for 3 minutes, adding equal volume of phenol chloroform (1: 1), shaking for several times by slow inversion, centrifuging at 12000rpm for 10 minutes, sucking supernatant of 100 microliters, washing with appropriate amount of 70% ethanol for 2 times, air drying, adding appropriate amount of ddH2O, storing in a refrigerator at-20 ℃.
2. Cloning PH04 gene, electrophoresis checking and sequencing verification.
And (3) PCR reaction system: 10 XExTaq PCR Buffer 5. mu.l, each of pH04-F/R primers 2. mu.l, dNTPmix (each 2.5mM) 2. mu.l, DNA 1. mu.l, enzyme 0.2. mu.l, ddH2O36.8. mu.l.
PH04-F primer: 5'-TTTCCAGCAAAGCGCCTCTT-3' are provided.
PH04-R primer: 5'-GAAGTCATGCTTCGGAAGGACC-3' are provided.
PCR reaction procedure: 85 ℃ 10s, 85 ℃ Pause plus enzyme Continue, 94 ℃ 3min, 35 cycles (94 ℃ 30s, 53.5 ℃ 30s, 72 ℃ 1min), 72 ℃ 10 min.
And (3) carrying out 0.8% agarose gel electrophoresis detection on the PCR product, carrying out sample sequencing after electrophoresis results (figure 1) and size verification are correct (the sizes are 1139bp because homology arms of 100bp are added below the upstream of the gene respectively), and comparing sequencing results to verify the correctness.
And 3, purifying the PCR product to obtain the PH04 gene.
And (3) recovering and purifying the PCR product of the PH04 gene by using a gel recovery kit, performing conventional operation according to the kit instruction, and detecting the purified product by 0.8% agarose gel electrophoresis at the temperature of-20 ℃ for storage in a refrigerator.
Example 2: obtaining recombinant high-yield yeast MF01-PH 04.
1. Original high-yield yeast spore-producing culture.
McClay spore-forming culture medium: 0.1% of glucose, 0.18% of potassium chloride, 0.82% of sodium acetate and 0.25% of yeast powder.
After yeast strains are activated, collecting seed liquid, washing thalli precipitates for 2 times by sterile water, inoculating the thalli precipitates into an McClay spore production culture medium, culturing at 28 ℃ for about 5 days, and detecting the spore production effect by a sampling microscope.
2. And preparing original high-yield yeast haploid protoplast.
Collecting spore-forming culture solution of the above 1, killing vegetative cells in water bath at 58 deg.C for 15min, washing with sterile water for 2 times, removing supernatant, re-suspending the precipitate with mixed enzyme solution (4% helicase, 2% cellulase), performing enzymolysis at 30 deg.C and 200rpm for 8h (every 1h after 4h), observing protoplast condition, and counting the number of protoplasts.
3. And (4) performing protoplast electrotransformation.
Controlling the number of protoplasts at 2 × 108Washing each ml with 0.7M potassium chloride once, centrifuging at 4 deg.C for 4000rpm for 5min, removing supernatant, washing with 1M sorbitol for 2 times (centrifuging at 4 deg.C for 4000rpm for 5min), removing supernatant, adding 1M sorbitol, adjusting the number of protoplast to 2 × 109Each ml is subpackaged into 1 ml sterile EP tube (80-90 microliter per tube), 10 microliter PH04 gene fragment is added, and after even mixing, the mixture is evenly mixedAfter the mixture was taken up in an electric rotary cup (pre-cooled in ice bath), it was subjected to ice-bath for 5min, and then to electric rotation for 1.5KV, 200. omega. 25. mu.F, 900. mu.l of 1M sorbitol was rapidly added (pre-cooled in ice bath).
4. And (4) regenerating the protoplast.
Regeneration culture medium: 2% of glucose, 2% of peptone, 1% of yeast powder and 5.22% of potassium chloride.
The transformation solution in the step 3 is evenly mixed, sucked out to a sterile 1.5mL EP tube, pre-cultured in water bath at 30 ℃ for 2h, added with 2mL regeneration medium, regenerated and cultured at 30 ℃ and 130rpm for 24h, and the regeneration condition is checked by microscopy.
5. And (3) verifying the recombinant high-yield yeast MF01-PH 04.
The culture solution in the above 4 was streaked on YPD plates, single colonies were randomly selected, DNA of colonies was extracted according to the method for extracting genomic DNA of low-yielding yeast in example 1, then the PH04 gene of candidate colonies was amplified according to the method for cloning the PH04 gene by PCR in example 1, PCR products were sent for sequencing, the sequencing results were compared, and it was verified that the original high-yielding yeast PH04 gene was replaced with the low-yielding yeast PH04 gene, thereby obtaining recombinant high-yielding yeast MF01-PH 04.
6. Stability of recombinant high-yield yeast MF01-PH 04.
After passage to 30 generations, the PH04 gene of 30 generations of recombinant yeast is amplified by referring to the method for cloning the PH04 gene by PCR in example 1, PCR products are sent for sequencing, sequencing results are compared, and the gene is found to be stable by sequencing.
Example 3: and (3) a cane molasses alcohol fermentation test of recombinant high-yield yeast MF01-PH 04.
1. The recombinant high-yield strain MF01-PH04 has a gas production difference with the original high-yield strain MF 01.
Activating the strains to logarithmic phase, counting microscopically, controlling the strains to be consistent, and taking the initial strains as 1 multiplied by 106The final concentration of each milliliter is inoculated into a test tube filled with an inverted Du's small tube (filled with YPD liquid culture medium) and 6mL of YPD, the test tube is placed into an incubator and cultured at the temperature of 30 ℃, and the gas production situation is observed every 1h after 4h, so that the recombinant high-yield strain MF01-PH04 produces gas faster than the original high-yield strain MF01 (see figure 2).
2. And (3) comparing the sugar cane molasses alcohol fermentation tests of the low-yield strain, the original high-yield strain and the recombinant high-yield strain.
The molasses alcohol fermentation is carried out according to the current production process of the cane molasses alcohol. Molasses culture medium: standard molasses 80-85 ° Bx from sugar mills. Molasses of 20-25 ℃ Bx, preferably 20 ℃ Bx, is used initially and molasses of 50-55 ℃ Bx, preferably 50 ℃ Bx, is used as feed. Both are weighed by water, the pH value is adjusted to 3.8-4.0 by sulfuric acid, and 0.2% (W/V) urea and 0.02% (W/V) phosphoric acid are added.
Activating strains to a logarithmic phase, adding saccharomyces cerevisiae with the inoculation amount of 5 percent into 20 DEG Bx molasses, culturing at 30 ℃ and 180rpm to form seed liquid, adding high-concentration molasses with the same volume of 50 DEG Bx, culturing for 24 hours, standing, fermenting, sampling every 8 hours to measure the alcohol content, fermenting for 32 hours, wherein the yield of the recombinant high-yield strain MF01-PH04 alcohol reaches the highest value of 114.71 grams per liter (56 hours), the highest value of the original high-yield strain alcohol is 108.94 grams per liter, the alcohol content of the former is improved by 5.3 percent compared with that of the latter, the highest value of the original high-yield strain is 112.58 grams per liter (64 hours) after 8 hours, and the alcohol yield of the low-yield strain is 86.35 grams per liter (64 hours), and the results are shown in figure 3.
And (3) determining the content of ethanol: the gas chromatograph is agilent 6890N using gas chromatography (internal standard method), conventional method.
The nucleotide sequence of the PH04 gene of the recombinant high-producing strain MF01-PH04 related by the embodiment is shown in a sequence table NO.1, and the amino acid sequence of the coded protein is shown in a sequence table NO. 2.
Claims (6)
1. A gene for promoting saccharomyces cerevisiae to perform high-concentration alcohol fermentation by utilizing cane molasses as a raw material is characterized in that the gene is PHO4, a positive regulation gene of a phosphorus system, and a nucleotide sequence of the gene is shown in a sequence table NO. 1.
2. The protein encoded by the gene of claim 1, wherein the amino acid sequence is as shown in sequence No. 2.
3. A strain of saccharomyces cerevisiae containing the gene of claim 1, characterized in that: the method is characterized in that a wild type saccharomyces cerevisiae strain with high yield of cane molasses alcohol is used as an initial strain, a PHO4 gene of the saccharomyces cerevisiae strain capable of rapidly growing but low yield of cane molasses alcohol is introduced into the initial strain, the introduced gene and the initial strain genome are subjected to homologous recombination, and the saccharomyces cerevisiae strain is obtained by screening.
4. The gene of claim 1, wherein the gene is derived from a strain MF01-PH04 capable of rapidly utilizing cane molasses as a raw material for high-concentration alcohol fermentation, the gene is classified under the name Saccharomyces cerevisiae and has been preserved in China center for type culture Collection, CCTCC for short, in 21.12.2018, with the preservation address of the preservation center of eight Wuhan university in flood mountainous areas of Wuhan city, Hubei province, and the preservation number of the gene is CCTCC M2018917.
5. The strain of claim 3 can be applied to the fermentation production of high-concentration alcohol of cane molasses, and is characterized in that: according to the fermentation process of the prior cane molasses alcohol production, 20-25 degrees of Bx molasses, preferably 20 degrees of Bx, is adopted, the pH value of sulfuric acid is adjusted to 3.8-4.0, after seed liquid is formed by culture, equal volume of 50-55 degrees of Bx molasses, preferably 50 degrees of Bx, is supplemented, the pH value of sulfuric acid is adjusted to 3.8-4.0, the cane molasses alcohol is fermented for 56 hours at 30 ℃, and the alcohol yield reaches the highest value of 114.71 grams per liter.
6. The strain of claim 3, wherein: the strain is an industrial saccharomyces cerevisiae strain.
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