CN105255951A - Method for improving ethyl alcohol production efficiency through HAC1 gene overexpression - Google Patents

Abstract

The invention discloses a method for improving alcohol production efficiency by overexpressing HAC1 gene, and belongs to the technical field of bioenergy development. In the present invention, the engineering strain obtained by overexpressing HAC1 in the ace2 single gene deletion strain is fermented to produce ethanol. Compared with the wild-type no-load control strain, the ethanol production is increased by 200.8%, and the yield of ethanol to bacteria is increased by 138.9%; Compared with the ace2 single gene deletion strain, the ethanol production is increased by 13.1%. It has good industrial application value and prospect, and provides important information for constructing an excellent Saccharomyces cerevisiae genetically engineered strain with high alcohol production.

Description

A method for improving ethanol production efficiency by overexpressing HAC1 gene

technical field

The invention relates to a method for improving ethanol production efficiency by overexpressing HAC1 gene, in particular to a method for improving the yield of ethanol to bacteria by using Saccharomyces cerevisiae with single gene deletion and overexpression of transcription factors, belonging to the technical field of bioenergy development.

Background technique

Fossil energy (coal, oil, natural gas, etc.) is currently the most important energy in the world. With the development of economy and society in various countries in the world, the demand for energy is increasing day by day, but the reserves of fossil energy are limited; at the same time, fossil energy The global warming caused by the greenhouse gases produced by combustion and the resulting problems have always troubled people. Therefore, searching for and developing renewable new energy sources has become a hot spot of concern all over the world. Fuel alcohol has the advantages of cleanness, safety, environmental friendliness, and renewable raw materials. It is considered to be an ideal substitute for petroleum resources and has a huge market potential. At present, it has been widely used in Europe, the United States, Brazil and other countries.

The production of fuel alcohol by microbial fermentation is a hot spot in current research. Saccharomyces cerevisiae is an ideal alcohol fermentation production strain, which has the following characteristics: it can grow and produce ethanol rapidly on a simple medium; and ethanol poisoning) have strong adaptability; genetic transformation is easy to operate; cheap substrates can be used, and the fermentation cost is low. In the past 10 years, the technology of producing ethanol by fermentation of thick mash has made great progress, but there is still a big gap compared with developed countries such as the United States. Osmotic pressure inhibition; toxicity inhibition caused by high concentration of ethanol in the later stage of fermentation; inhibition caused by uncontrollable high temperature during fermentation. The existence of these problems has become the main factor that limits the production efficiency of thick mash fermentation technology.

At present, a lot of progress has been made in the research on the tolerance of Saccharomyces cerevisae to high osmotic pressure, high concentration of alcohol and high temperature, but the related tolerance mechanism has not been clearly studied. Therefore, it is very difficult to improve the tolerance of S. cerevisiae to adversity through rational transformation. Although the traditional methods of strain selection (such as natural screening, mutation breeding) have achieved certain effects in improving the stress tolerance of strains, there are disadvantages such as heavy workload and randomness. In recent years, with the rapid development of molecular biology and omics technology, conditions have been provided for large-scale research on the relationship between Saccharomyces cerevisiae genes and ethanol fermentation.

Contents of the invention

The first technical problem to be solved by the present invention is to provide a method for improving ethanol production efficiency. The method is to overexpress the HAC1 gene in the ace2 gene deletion strain to improve the ethanol production efficiency of yeast fermentation.

In one embodiment of the present invention, the engineered bacterium is an engineered bacterium of Saccharomyces cerevisiae.

In one embodiment of the present invention, the nucleotide sequence of the ACE2 gene is shown in SEQ ID NO.1.

In one embodiment of the present invention, the nucleotide sequence of the HAC1 gene is shown in SEQ ID NO.2.

In one embodiment of the present invention, the amino acid sequence encoded by the ACE2 gene is shown in SEQ ID NO.3.

In one embodiment of the present invention, the method is to overexpress the HAC1 gene in Saccharomyces cerevisiae with single gene deletion of ace2.

In one embodiment of the present invention, the Saccharomyces cerevisiae with single gene deletion of ace2 is purchased from Invotrogen Company, the number is Sc04146899_s1.

In one embodiment of the present invention, the overexpression is to clone the HAC1 gene fragment into the expression plasmid pHAC181 (JiangL, 2004) to obtain the recombinant high-expression plasmid pHAC181-HAC1, and then transform the recombinant plasmid into the ace2 single gene deletion strain express in.

在本发明的一种实施方式中，所述表达质粒pHAC181是在YCplac181的SphI和EcoRV位点插入3个组氨酸标签得到的，详见文献：AnalysesoftheeffectsofRck2pmutantsonPbs2p ^DD -inducedtoxicityinSaccharomycescervisiaeidentifyaMAPkinasedockingmotif,andunexpectedfunctionalinactivationduetoacidicsubstitutionofT379,MolGenGenomics,2004,271 :208–219.

In one embodiment of the present invention, the fermentation production is to transfer the yeast engineered bacteria to the fermentation medium after activation, so that the OD ₆₀₀ of the fermentation medium after inoculation of the seed bacteria liquid is 0.4-0.5, and the fermentation medium is heated at 28-30°C. , 200-220r/min culture for 7-8 hours and then transfer to static culture, static culture for 50-52h.

In one embodiment of the present invention, the fermentation production is to transfer the yeast engineered bacteria to the fermentation medium after activation, so that the _OD600 of the fermentation medium after inoculation of the seed bacteria liquid is 0.5, and the fermentation is carried out at 30°C and 220r/min After culturing for 8 hours, transfer to static culture and continue culturing for 52 hours.

In one embodiment of the present invention, the fermentation medium contains 100 g/L of glucose, 7.5 g/L of ammonium sulfate, 3.5 g/L of potassium dihydrogen phosphate, 0.75 g/L of magnesium sulfate heptahydrate, and 0.2 g/L of yeast extract. g/L, histidine 0.02g/L, uracil 0.02g/L, leucine 0.1g/L.

In one embodiment of the present invention, the activation is preferably YPD medium. Streak on the YPD solid medium, culture at 30°C for 2 days to preliminarily activate the strains, then transfer to YPD liquid medium, culture at 30°C, 220r/min shaker for 23h to saturation.

The second technical problem to be solved by the present invention is to provide a Saccharomyces cerevisiae engineered bacterium with improved ethanol production efficiency. The Saccharomyces cerevisiae engineered bacterium has deleted the ace2 gene encoding the amino acid sequence such as SEQ ID NO.3 and overexpressed the nucleotide sequence such as HAC1 gene of SEQ ID NO.2.

The Saccharomyces cerevisiae engineered bacteria overexpresses the HAC1 gene in Saccharomyces cerevisiae with single gene deletion of ace2.

Beneficial effects of the present invention:

The present invention utilizes the engineered yeast strain obtained by overexpressing the HAC1 gene in the Saccharomyces cerevisiae strain with single gene deletion of ace2 to ferment and produce alcohol. Compared with the wild-type no-load control strain, when the fermentation is carried out for 52 hours, the ethanol production is increased by 200.8%. The yield of ethanol to the bacteria increased by 138.9%; compared with the strain with only ace2 single gene deletion, the ethanol production increased by 13.1%. The invention has good industrial application value and prospect, and at the same time provides important information for constructing an excellent Saccharomyces cerevisiae genetically engineered strain with high alcohol production.

Description of drawings

Figure 1: Comparison of bacterial biomass; where ace2+HAC1 represents the engineering strain obtained by overexpressing HAC1 in the ace2 single gene deletion strain, and WT+V represents the wild-type control strain containing no load;

Figure 2: Comparison of ethanol production; where ace2+HAC1 represents the engineering strain obtained by overexpressing HAC1 in the ace2 single gene deletion strain, and WT+V represents the wild-type control strain containing no load;

Figure 3: Comparison chart of ethanol yield; where ace2+HAC1 represents the engineering strain obtained by overexpressing HAC1 in the ace2 single gene deletion strain, and WT+V represents the wild-type control strain containing no load.

detailed description

The method for the determination of ethanol content by high performance liquid chromatography: the determination of ethanol content in the fermentation broth is detected by high performance liquid chromatography (HPLC). After the fermentation broth is treated and the supernatant is filtered through a 0.22 μm microporous membrane, it is detected by RID (refractive index detector). The liquid chromatography method is as follows: Chromatographic column: Shodex SH1011 sugar column organic acid column; ; Mobile phase: 0.0275% (v/v) dilute sulfuric acid, filtered through a 0.22 μm filter membrane and degassed; flow rate: 1 mL/min; detection time: 17 min; injection volume: 20 μL.

Calculation method of ethanol to thallus yield: the yield calculation formula is as follows:

$\mathrm{<span\; class="notranslate">\; the\; y</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; p</span>}}{\mathrm{<span\; class="notranslate">\; x</span>}}$

In the formula, y is the yield of ethanol, p is the concentration of ethanol, X is the dry weight of cells x=0.2Z, and Z is the OD value.

Composition of YPD medium: 2% glucose, 1% yeast extract, 2% peptone, deionized water, natural pH, autoclaved (115° C., 20 min).

The composition of the fermentation medium (g/L): glucose 100, ammonium sulfate 7.5, potassium dihydrogen phosphate 3.5, magnesium sulfate heptahydrate 0.75, yeast extract 0.2, histidine 0.02, uracil 0.02, leucine 0.1, de Dilute to volume with ionized water, keep the pH natural, and sterilize under high pressure (115°C, 20min).

Embodiment 1: Construction of Saccharomyces cerevisiae Genetic Engineering Bacteria

With the ace2 single gene deletion strain YLR131C (http://www.lifetechnologies.com/order/genome-database/browse/gene-expression/keyword/YLR131C?ICID=search-gex-YLR131C purchased from Invotrogen Company, No. Sc04146899_s1 ) as the starting strain, overexpression is by constructing the high-expression plasmid pHAC181-HAC1, the specific method is the HAC1 gene fragment (including 865bp promoter sequence and its ORF frame, excluding stop codon, a total of 1850bp), and then clone this fragment On the high-expression plasmid pHAC181, DNA sequencing was performed on the correct recombinant, and no mutations occurred in the sequence, and finally the recombinant high-expression plasmid pHAC181-HAC1 was obtained, and then the plasmid pHAC181-HAC1 was introduced into the ace2 single gene deletion strain by the lithium acetate method. Thus, a strain with high expression of HAC1 gene was obtained.

Example 2: Ethanol fermentation by Saccharomyces cerevisiae

Prepare YPD plate and YPD liquid medium in advance according to the preparation method of solid medium.

Streak activation of Saccharomyces cerevisiae genetically engineered bacteria of the present invention, wild-type strain BG (BY4743 containing G418 resistance gene) (http://clones.lifetechnologies.com/cloneinfo.php?clone=yeast) and ace2 on the YPD plate Saccharomyces cerevisiae strain YLR131C with a single gene deletion (the ace2 single gene deletion strain was obtained by replacing the ACE2 gene with the G418 resistance gene in the BY4743 background strain). The YPD plate was placed in a constant temperature incubator at 30°C for 2 days. Working in an ultra-clean bench, take a large single colony from each activated strain plate and inoculate it in 30 mL of YPD liquid medium, culture it on a shaker at 220 r/min at 30°C for 23 hours until saturated. After culturing for 23 hours, add 50 μL of overnight cultured strain bacterial solution to the EP tube, dilute it 20 times, and measure the OD ₆₀₀ value. According to the measured OD ₆₀₀ value, calculate the amount of added seed bacteria solution, so that the final OD ₆₀₀ value of the fermentation medium after inoculating the seed bacteria solution is 0.5, and add the calculated amount of seed bacteria solution to 100mL on the ultra-clean bench in the fermentation medium.

Put the fermentation medium into 30°C, 220r/min shaker culture, and transfer to static culture after 8 hours.

At the 32h and 52h fermentation time points, take 1000μL of the bacterial liquid in a 1.5mL EP tube, centrifuge in a centrifuge at 12000rpm for 4min, and transfer 600μL of the supernatant to two groups of 1.5mL EP tubes after centrifugation. One group was added with 600 μL trichloroacetic acid and stored overnight in a refrigerator at 4°C, and the concentration of ethanol was measured by liquid phase the next day. The other group was stored in a refrigerator at -20°C without adding trichloroacetic acid.

At the same time, at the 32h and 52h fermentation time points, three groups of 50 μL fermentation broth samples were taken, diluted 20 times in a 1.5mL EP tube, the OD value was measured, and the data was recorded for calculation of bacterial biomass.

The first group of supernatant samples stored in a 4°C refrigerator were centrifuged at 12,000 rpm for 2 min at room temperature, and 600 μL of the supernatant was filtered, and the filtrate was transferred to a liquid phase sample bottle to measure the ethanol concentration. Samples that cannot be arranged for liquid phase measurement immediately should be stored in a refrigerator at -20°C.

Take the liquid phase sample to be tested, apply the liquid phase instrument, copy the test data, make the data into an Excel table, and draw the OD, ethanol production, and ethanol production rate graphs, as shown in Figure 1-3, and apply it to winemaking with ace2 gene deletion The engineered yeast strain obtained by overexpressing the HAC1 gene in the yeast strain produced alcohol by fermentation. Compared with the wild-type empty control strain (that is, the wild-type strain BG containing the empty plasmid pHAC181, represented by WT+V), the fermentation proceeded to 52h , the ethanol production increased by 200.8%, and the yield of ethanol to bacteria increased by 138.9%. Compared with the ace2 single gene deletion strain, the ethanol production increased by 13.1%. In addition, the present invention also found that the ethanol production efficiency of the yeast engineered bacteria that individually deleted the HAC1 gene was reduced.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. improve a method for Alcohol Production efficiency, it is characterized in that, described method utilizes ace2 genetically deficient and the Yeast engineering bacteria fermentative production alcohol of overexpression HAC1 gene.

2. method according to claim 1, is characterized in that, the aminoacid sequence of described ace2 genes encoding is as shown in SEQIDNO.3.

3. method according to claim 1, is characterized in that, the nucleotide sequence of described HAC1 gene is as shown in SEQIDNO.2.

4. according to the arbitrary described method of claim 1-3, it is characterized in that, described method is process LAN HAC1 gene in the yeast saccharomyces cerevisiae of ace2 single-gene disappearance.

5. method according to claim 1, is characterized in that, described fermentative production is forwarded to fermention medium after being activated by Yeast engineering bacteria, makes to inoculate kind of an OD for daughter bacteria liquid post-fermentation and culture base ₆₀₀for 0.4-0.5, in 28-30 DEG C, 200-220r/min cultivates after 7-8 hour and transfers quiescent culture to, quiescent culture 50-52h.

6. method according to claim 5, is characterized in that, described fermentative production is forwarded to fermention medium after being activated by Yeast engineering bacteria, makes to inoculate kind of an OD for daughter bacteria liquid post-fermentation and culture base ₆₀₀be 0.5, in 30 DEG C, 220r/min transfers quiescent culture to after cultivating 8 hours, continue to cultivate 52h.

7. the method according to claim 5 or 6, it is characterized in that, described fermention medium contains glucose 100g/L, ammonium sulfate 7.5g/L, potassium primary phosphate 3.5g/L, magnesium sulfate heptahydrate 0.75g/L, yeast extract 0.2g/L, Histidine 0.02g/L, uridylic 0.02g/L, leucine 0.1g/L.

8. a saccharomyces cerevisiae engineered yeast, is characterized in that, described saccharomyces cerevisiae engineered yeast lacked encoding amino acid sequence as the ace2 gene of SEQIDNO.3 and process LAN nucleotide sequence as the HAC1 gene of SEQIDNO.2.

9. saccharomyces cerevisiae engineered yeast according to claim 8, is characterized in that, described saccharomyces cerevisiae engineered yeast is process LAN HAC1 gene in the yeast saccharomyces cerevisiae of ace2 single-gene disappearance.

10. the application of saccharomyces cerevisiae engineered yeast described in claim 8 or 9 in Alcohol Production.