CN106434700A - Saccharomyces cerevisiae spt15 fixed point saturated gene mutation method for increasing yield of ethanol - Google Patents

Abstract

The invention proposes a point-saturation gene mutation method of Saccharomyces cerevisiae spt15 for improving ethanol yield. It uses genetic engineering to obtain the spt15 site-directed saturation mutant gene of Saccharomyces cerevisiae, and connects it to the expression vector pYES2NTc to construct a mutation library. A total of 12 single-point mutant genes have been obtained, which are compatible with wild-type genes spt15, spt3 and the control gene Neo gene. Recombinant plasmids were constructed and transformed into Saccharomyces cerevisiae INVSC1 by lithium acetate method to obtain 15 recombinant Saccharomyces cerevisiae strains. Using glucose as a substrate, the recombinant Saccharomyces cerevisiae was inoculated and fermented, and the ethanol production rate was measured. Among them, the ethanol production rate of the recombinant Saccharomyces cerevisiae INVSC1‑SPT15‑M and INVSC1‑SPT15‑N was greatly improved, respectively 43.0±0.9g/ L and 43.7±0.2g/L. Compared with the control strain INVSC1‑Neo, it increased by 17.8% and 19.7%, respectively. Two mutant genes were mutated from lysine to methionine and asparagine at position 127 respectively. The method of the invention is simple, easy to operate, and has an obvious effect of improving the yield of ethanol. It has good economic and social benefits.

Description

A method for site-directed saturation gene mutation of Saccharomyces cerevisiae spt15 to increase ethanol yield

technical field

The present invention relates to the technology of biomass fermentation to produce ethanol, especially a kind of Saccharomyces cerevisiae spt15 fixed-point saturation gene mutation method for improving ethanol yield

Background technique

Traditional bioethanol production is mainly based on grain fermentation, and the world's food shortage severely limits the source of raw materials. Lignocellulose is the most abundant biomass resource in the world, with the largest amount and the cheapest price. The total annual output accounts for about 50% of all biomass resources ^[1] , but most of these substances are not used efficiently. As a clean and renewable energy, industrial ethanol produced from biomass is an inevitable trend to replace fossil fuels such as petroleum, and has been included in the development strategy planning of many countries.

Saccharomyces cerevisiae (S.cerevisiae) is a eukaryotic microorganism with thick cell wall and high sterol content, and has a high tolerance to toxic factors in ethanol and lignocellulose hydrolyzate[3]; it can survive under low pH and strict anaerobic conditions Under the rapid fermentation of glucose to produce ethanol, there are few by-products; it is not easy to be polluted by bacteria and viruses, and the related industrial technology is mature. The whole genome sequencing of Saccharomyces cerevisiae has been completed[4], which is the most thoroughly studied eukaryotic microorganism so far. The methods and means of bioinformatics and the increasingly perfect molecular biology techniques can be used to carry out genetic manipulation and metabolic network analysis on it. refactor. It is the research focus and primary target microorganism of ethanol fermentation. The ethanol tolerance of yeast is related to multiple genes, so it is difficult to improve the ethanol tolerance and ethanol yield of yeast through traditional single gene knockout or overexpression. In 2006, Alper et al. reported the groundbreaking research on improving the ethanol yield and tolerance of yeast by gTME method, which provided a new idea for the metabolic engineering operation of ethanol yield and tolerance of yeast.

Transcription level regulation is the most efficient part of gene expression regulation. Global transcription machinery engineering (gTME) is a technique to optimize cell phenotype through gene transcriptional rearrangement ^[6] , which uses molecular biology methods such as error-prone PCR, DNA shuffling, etc. to establish the initial Transcription factor mutation library, modify global transcriptional regulatory factors, conduct directional screening for target products or target phenotypes, change the entire transcriptional regulation process to change or improve the transcription and expression of target genes, and obtain target metabolic flux enhancement or specific phenotype enhancement strains. Transcription is performed by RNA polymerase. In eukaryotes, RNA polymerase II is responsible for the transcription of mRNA that produces most functional genes, and the transcription efficiency of RNA polymerase II is determined by the binding ability of the initial transcription factor and the promoter. spt15, one of the initiation transcription factors in Saccharomyces cerevisiae, belongs to the transcription initiation complex, and is a TATA-binding protein ^[7] , which participates in the formation of the transcription initiation complex and controls gene expression efficiency. Changes in its ability to bind to TATA in the promoter region of related genes affect the expression efficiency of related genes, thereby causing phenotypic changes in the strain. Its mutation overexpressed related genes, and phenotypically improved the tolerance of yeast to ethanol.

Chinese patent: A mutant Saccharomyces cerevisiae initiation transcription factor and its coding gene and application (ZL200810024036.8) It uses random mutation technology to mutate the transcription factor spt15 gene, and obtains a mutant spt15-6 of spt15 after screening; another Chinese patent Patent: Mutated Saccharomyces cerevisiae initiation transcription factor gene and its expression vector and application (ZL201310008539.7) also uses random mutation technology to mutate the transcription factor spt15 gene, and obtains a mutant spt15-10 of spt15 after screening. Alignment of them with the spt15 gene sequence of Saccharomyces cerevisiae showed obvious changes. Using genetic engineering technology to construct recombinant Saccharomyces cerevisiae, the yield of ethanol in the fermentation test has changed, but it is not ideal for engineering applications, that is, the yield of ethanol should be further improved.

The present invention amplifies the initial transcription regulator gene spt15 from the wild-type yeast strain, obtains the mutant gene by the site-directed saturation mutation method, constructs it into the pYES2NTc vector, and transforms it into Saccharomyces cerevisiae INVSC1 for expression through the lithium acetate method. Yeast ethanol production was measured. Two point mutations of spt15 gene were obtained after screening. The metabolic properties of the recombinant Saccharomyces cerevisiae recombined after mutation are greatly improved, that is, the ethanol production rate thereof is greatly improved.

references

[1] Liu HM, Xu L, Yan M, et al. gTME for construction of recombinant yeastco-fermenting xylose and glucose. Chin J Biotech, 2008, 24(6): 1 6.

Liu Hongmei, Xu Lin, Yan Ming, et al. Construction of recombinant Saccharomyces cerevisiae co-fermenting xylose and glucose with gTME. Chinese Journal of Bioengineering, 2008,24(6):1 6.

[2] Hal A, Gregory S. Global transcription machinery engineering: A new approach for improving cellular phenotype. MetabEng, 2007, 9: 258 267.

Contents of the invention

The invention proposes a method for mutating the spt15 fixed-point saturation gene of Saccharomyces cerevisiae by means of genetic engineering to improve the ethanol yield.

The present invention is achieved like this. A method for site-directed saturation gene mutation of Saccharomyces cerevisiae spt15 for improving ethanol yield, the steps of which are:

1. Amplification of S.cerevisiaeINVSC1spt15 gene and construction of recombinant plasmid

a) Using a genome extraction kit, extract the S.cerevisiaeINVSC1 genome. Using the total DNA of S. cerevisiae as a template, the Spt15_Forward and Spt15_Reverse primers were used to amplify the Saccharomyces cerevisiae initiation transcription factor spt15 gene by PCR. The PCR cycle parameters are: 94°C, 5min; 94°C for 1min, 56°C for 1min, 72°C for 2min, 30 cycles. The amplified gene was purified and recovered with a gel recovery kit (TaKaRa Company).

Primer: Spt15_Forward5'-ATGGCCGATGAGGAACGTTTAAAGGAGTTTA-3'

Spt15_Reverse5'-TCACATTTTTCTAAATTCACTTAGCACAGGGTATATAG-3'

The original spt15 gene sequence is shown in the sequence listing SEQ ID No.1

b) The recovered spt15 gene was ligated with the pMD18-T (TaKaRa company) vector, and the ligated vector was transformed into Escherichia coli DH5α competent cells by the calcium chloride method, and the monoclonal recombinant plasmid pMD18-spt15 was extracted, and the gene sequence spt15 was obtained by sequencing. . The spt15 gene on the pMD18-spt15 plasmid was then PCR amplified using the Spt15_yes2ntF and Spt15_yes2ntR primers. The PCR cycle parameters are: 94°C, 5min; 94°C for 1min, 56°C for 1min, 72°C for 2min, 30 cycles. The amplified gene was purified and recovered with a gel extraction kit (TaKaRa Company) after NotI and BamHI double enzyme digestion.

Primer: Spt15_yes2ntF5'-GGATCCGCCGATGAGGAACGTTTA-3'

Spt15_yes2ntR5'-GCGGCCGCTCACATTTTTCTAAATTCAC-3'

c) The recovered spt15 gene was ligated with the pYES2NTc linear vector that had been digested and recovered by NotⅠ and BamHI, and the ligated vector was transformed into Escherichia coli DH5α competent cells by the calcium chloride method, and the monoclonal recombinant plasmid pYES2NTc-spt15 was extracted. The gene sequence spt15 obtained by sequencing was correct.

2. Site-directed saturation mutation gene library and acquisition of recombinant Saccharomyces cerevisiae

a) Through the homologous comparison and three-dimensional structure analysis of spt15 sequence, it is found that the Lys127 site is located in the middle binding site of the symmetrical structure. The mutation of this site may affect the binding of spt15 and spt3, so this site is selected as the mutation site .

b) According to the principle of reverse overlap extension PCR (overlap extension PCR), design a pair of mutation primers Spt15_mutF and Spt15_mutR, where the lowercase underlined part is the mutation site, corresponding to the 127 amino acid codon.

First, the recombinant plasmid pYES2NTc-spt15 was used as a template, and the primers Spt15_mutF and Spt15_mutR were used for plasmid amplification. The PCR amplification cycle parameters were 94 ° C for 50 sec, 62 ° C for 45 sec, 72 ° C for 7 min, 30 cycles, and finally, 72 ° C for 10 min. Reverse amplification to obtain a linear fragment containing the vector sequence and gene sequence. After digesting the template with DpnI enzyme, the gel was recovered, self-ligated, transformed into E. coli DH5α, and the transformant was screened on a kanamycin resistance plate, and sequenced to identify whether it was a mutant gene . The comparison of the mutant gene sequence with the original Saccharomyces cerevisiae spt15 gene sequence ^[10] showed that a total of 12 different point mutations at the 127th lysine were obtained, and no mutations occurred at other sites. The results of the 12 point mutations are shown in Table 1.

Primer: Spt15_mutF5'-ATGGTTGTTACCGGTGCA nnk AGTGAGGATGACTCA-3'

Spt15_mutR5'-TGCACCGGTAACAACCATTTTCCCTGAGGCAAAAATTAAAGC-3'

c) The recombinant plasmids containing the mutated and unmutated spt15 genes were transformed into Saccharomyces cerevisiae INVSC1 by the lithium acetate method ^[9] . Use the SX screening medium to screen transformants to obtain a series of recombinant Saccharomyces cerevisiae (12 in total) containing the spt15 mutant gene, which are respectively named as a series of recombinant Saccharomyces cerevisiae INVSC1-spt15-X according to the sequence number of the mutant gene, wherein X is F, V, R, M, L, G, T, S, Q, D, N, I (see Table 1).

Table 1 Codons and amino acids of site-directed saturation mutation sites

3. Fermentation experiment, result analysis and screening of recombinant Saccharomyces cerevisiae INVSC1-spt15-X.

Inoculate the 12 recombinant Saccharomyces cerevisiae obtained above and the control group into 50mL seed medium respectively, culture them at 30°C and 200r/min for 24h, and inoculate them in a 500mL Erlenmeyer flask containing 100mL fermentation medium at 10% (V/V) After appropriate dilution of the bacterial solution, the absorbance value was measured at 600nm, and the selected strains in the logarithmic growth phase were inoculated into 100mL fermentation medium for anaerobic fermentation at 30°C and 200r/min. Determination of ethanol content and residual sugar content. The fermentation broth samples were filtered through a 0.45 μm cellulose acetate filter membrane, and the ethanol concentration and glucose concentration were detected using a SBA-40C biosensor analyzer and reagents (Institute of Biology, Shandong Academy of Sciences). After determination, analysis and screening, the recombinant bacteria recombinant Saccharomyces cerevisiae INVSC1-SPT15-M, INVSC1-SPT15-N, the production rate of ethanol using glucose has been greatly improved, and the ethanol production rate in the glucose medium is 43.0±0.9g/ L and 43.7±0.2g/L. They were 17.8% and 19.7% higher than the control strain, respectively.

The preferred spt15-M mutant gene sequence is shown in SEQ ID No.2, and the mutant gene is mutated from lysine to methionine at position 127.

The preferred spt15-N mutant gene sequence is shown in SEQ ID No. 3, and the mutant gene is mutated from lysine to asparagine at position 127 respectively.

The principle of the present invention is like this. The phenotype of a cell is determined comprehensively by many genes, and the construction of a strain with an ideal phenotype requires simultaneous multi-gene modification, but the ability to introduce these modifications is usually very limited. Gene expression regulation occurs at various levels of genetic information transmission, and transcription regulation is the most important link in gene expression regulation. A global transcriptional machinery engineering approach allows altering the expression of many terminal genes. That is, by changing the response to the transcriptional protein, the entire transcript is greatly disturbed. By modifying the initial transcriptional regulator spt15 and using specific screening conditions to obtain an optimized target phenotype, it is through the change of the initial transcription factor to realize the simultaneous change of multiple genes to regulate the entire metabolic network.

The present invention selects the Lys127 site as the mutation point to carry out fixed-point saturation mutation, and obtains obvious technical effects.

The invention has clear advantages. The method of the invention is simple, easy to operate, and has an obvious effect of improving the yield of ethanol. It has good economic and social benefits.

Description of drawings

Figure 1. Cloning of spt15 gene and electrophoresis detection verification of pYES2NTc-spt15 recombinant plasmid

Wherein (A) 1: PCR product of spt15; M: DNA marker DL2000

(B) 1: Double enzyme digestion verification of pMD18-spt15 plasmid; M: DNA marker DL10000

(C) 1: PCR verification of pYES2NTc-spt15 plasmid; 2: pYES2NTc-spt15 plasmid;

3: pYES2NTc-spt15 single digestion product; M: DNA marker DL5000

Figure 2 Sugar utilization of the mutant recombinant yeast INVSC1-spt15-X

Figure 3 Ethanol production rate of mutant gene recombinant yeast INVSC1-spt15-X and control strain

Figure 4 The ethanol production and glucose utilization curves of the mutant gene recombinant yeast strain INVSC1-SPT15-M, INVSC1-SPT15-N and the control strain recombinant strain INVSC1-G418;

Figure 5 The three-dimensional structure and mutation site of the initiation transcription regulator gene spt15 in the wild-type yeast strain;

detailed description

Below with example the present invention is further described:

Example 1: Cloning of the spt15 gene and construction of its recombinant plasmid

Using the genomic DNA of S. cerevisiaeINVSC1 as a template, the designed primers were used for PCR reaction, and there was an obvious band at about 0.75kb in the electrophoresis detection of the PCR product (Figure 1). After purification and recovery, the PCR product was ligated with the cloning vector pMD18-T, and the ligated product was transformed into Escherichia coli E.coli DH5α, and the positive transformant pMD18-spt15 was screened by blue and white spots. The results of plasmid sequencing showed that the obtained spt15 sequence was correct, and the gene bank The homology of the gene sequence is 100% (GenBank gene number M29459.1, protein number AAA34458.1).

It was amplified by PCR on the plasmid pMD18-spt15, digested with NotI and BamHI double enzymes, recovered and purified by agarose gel electrophoresis. The recovered spt15 gene was ligated with the pYES2NTc linear vector that had been digested and recovered by NotI and BamHI, and the recombinant plasmid pYES2NTc-spt15 was obtained after PCR and enzyme digestion verification, and the gene sequence spt15 was obtained by sequencing.

Example 2 Obtaining of Site-directed Saturation Mutation Gene Library and Recombinant Saccharomyces cerevisiae

Using the recombinant plasmid pYES2NTc-spt15 obtained in Example 1 as a template, carry out reverse overlap extension PCR, digest with DpnI enzyme, recover and self-ligate, transform Escherichia coli DH5α, screen transformants on a kanamycin resistance plate, and identify whether they are for the mutant gene. The comparison of the mutant gene sequence with the original Saccharomyces cerevisiae spt15 gene sequence ^[10] showed that a total of 12 different point mutations at the 127th lysine were obtained, and no mutations occurred at other sites. The results of the 12 point mutations are shown in Table 1.

Example 3 Screening of Mutant Gene Recombinant Saccharomyces cerevisiae

The recombinant plasmids containing mutated and unmutated spt15 genes were transformed into Saccharomyces cerevisiae INVSC1 by lithium acetate method ^[9] . Transformants were selected using a selection medium lacking uracil, resulting in a series of recombinant S. cerevisiae containing the spt15 mutant gene. Named as a series of recombinant Saccharomyces cerevisiae INVSC1-spt15-X according to the sequence number of the mutant gene, where X is K, F, V, R, M, L, G, T, S, Q, D, N, I (see Table 1) . Where K is not mutated.

Example 4 Sugar Utilization of INVSC1-spt15-X Mutant Gene Recombinant Yeast

Most genes showed varying degrees of reduction in glucose consumption rate. Especially SPT15-N, SPT15-K. However, both the control strain and the mutant gene recombined strain consumed glucose completely within 12 hours, indicating that the glucose consumption rate did not affect their ethanol production. (See Figure 3)

Example 5 Detection of Mutant Gene Recombinant Saccharomyces INVSC1-spt15-X Anaerobic Fermentation Ethanol Production

Table 2 shows the ethanol yields of the control strain INVSC1-G418 and the recombinant strain INVSC1-spt15-X fermented in 100 g/L glucose medium at 30°C and 200 r/min for 48 hours.

Compared with the control strain INVSC1-G418, the ethanol production of INVSC1-SPT15-L, INVSC1-SPT15-G, INVSC1-SPT15-K, INVSC1-SPT15-I, INVSC1-SPT15-S basically did not change, and the highest peaks were all at 24h. While INVSC1-SPT15-T, INVSC1-Spt3, INVSC1-SPT15-Q, INVSC1-SPT15-F, INVSC1-SPT15-V, INVSC1-SPT15-D ethanol yield decreased significantly, and the ethanol yield was around 25-29g/L . It is 68.5%-79.5% of the control strain. Although the highest ethanol production rate of SPT15-R did not change much compared with the control bacteria, the time to reach the highest ethanol production was delayed from 24h to 48h.

Recombinant yeasts INVSC1-SPT15-M and INVSC1-SPT15-N have greatly improved ethanol production rates using glucose, and the ethanol production rates in glucose medium are 43.0±0.9g/L and 43.7±0.2g/L respectively . They were 17.8% and 19.7% higher than the control strain, respectively. The results preliminarily reveal that the effective mutation of the initiation transcription factor spt15 gene has significantly changed the metabolic pathway and metabolic flow of Saccharomyces cerevisiae.

Table 2 Comparison of ethanol production between recombinant bacteria and control strains under the same conditions

Description of materials used in the present invention.

Escherichia coli DH5, host strain of Saccharomyces cerevisiae INVSC1, and yeast expression vector pYES2NTc can all be purchased.

The genomic DNA extraction kit used in the experiment was purchased from Shanghai Huashun Bioengineering Co., Ltd., the restriction endonuclease NotⅠ and BamHI were purchased from NEB Company, and the pMD18-T vector and gel recovery kit were produced by Dalian Bao Biological Engineering Co., Ltd. (TaKaRa) , primer synthesis, plasmid extraction kit, Taq polymerase, dNTP Mixture ampicillin antibiotics were produced by Shanghai Sangon Bioengineering Co., Ltd., TransTaq DNA Polymerase High Fidelity high-fidelity PCR polymerase was produced by Quanshijin Company, and the gene sequence was determined by Yingjun (Invitrogen) Biological Limited completed. Other reagents are analytically pure.

culture medium

Escherichia coli was cultured with LB medium, supplemented with 50 μg/mL ampicillin. Saccharomyces cerevisiae was cultured on YPAD medium.

Basic medium (YPAD) (g/L): yeast powder 10, peptone 20, glucose 20, adenine sulfate 0.075.

Screening medium (SX) (g/L): amino acid-free yeast nitrogen source (YNB) 6.7, essential amino acid mixture (lacking uracil) 1.3, glucose 20, agar powder 20.

Seed medium (g/L): yeast powder 10, peptone 20, glucose 20.

Fermentation medium (g/L): yeast powder 10, peptone 20, glucose 100.

Claims (8)

1. a Saccharomyces cerevisiae spt15 site-directed saturation mutant gene that improves ethanol production rate is characterized in that its sequence is the spt15-M mutant gene shown in SEQID No.2, and this mutant gene is mutated into formazan by lysine at 127 positions thionine. 2. A Saccharomyces cerevisiae spt15 site-directed saturation mutant gene that improves ethanol production rate is characterized in that its sequence is the spt15-N mutant gene shown in SEQID No.3, and this mutant gene is mutated into asparagus by lysine at position 127 amides. 3. A recombinant expression vector of Saccharomyces cerevisiae spt15 site-directed saturation mutation gene for improving ethanol yield, characterized in that said recombinant expression vector contains the spt15-M mutant gene whose sequence is shown in SEQ ID No.2. 4. A recombinant expression vector of Saccharomyces cerevisiae spt15 site-directed saturation mutation gene for improving ethanol yield, characterized in that the recombinant expression vector contains the spt15-N mutant gene whose sequence is shown in SEQ ID No.3. 5. A recombinant Saccharomyces cerevisiae that improves ethanol production rate Saccharomyces cerevisiae spt15 site-directed saturation mutation gene, characterized in that said recombinant Saccharomyces cerevisiae contains the spt15-M mutant gene whose sequence is shown in SEQ ID No.2, named as INVSC1- SPT15-M. 6. A recombinant Saccharomyces cerevisiae that improves the ethanol production rate of Saccharomyces cerevisiae spt15 site-directed saturation mutation gene, characterized in that the recombinant Saccharomyces cerevisiae contains the spt15-N mutant gene whose sequence is shown in SEQ ID No.3, named INVSC1- SPT15-N. 7. An application method of a recombinant Saccharomyces cerevisiae spt15 site-directed saturation mutation gene improving ethanol productivity, characterized in that the recombinant Saccharomyces cerevisiae described in claim 5 is used to use glucose as a substrate for fermentation to produce ethanol, ethanol production The rate can reach 43.0±0.9g/L, which is 17.8% higher than that of the control strain. 8. An application method of a recombinant Saccharomyces cerevisiae spt15 site-directed saturation mutation gene improving ethanol production rate, characterized in that the recombinant Saccharomyces cerevisiae according to claim 6 is used to produce ethanol by fermentation with glucose as a substrate, and the ethanol production The rate can reach 43.7±0.2g/L, which is 19.7% higher than that of the control strain.