CN109825516B

CN109825516B - Saccharomyces cerevisiae site-directed saturation mutant gene spt15-N for improving ethanol yield and application thereof

Info

Publication number: CN109825516B
Application number: CN201910195929.7A
Authority: CN
Inventors: 柯涛; 赵珊珊; 吴时玺; 姜鹏; 闫沛喆; 徐树林
Original assignee: Nanyang Normal University
Current assignee: Nanyang Normal University
Priority date: 2016-08-19
Filing date: 2016-08-19
Publication date: 2020-01-14
Anticipated expiration: 2036-08-19
Also published as: CN106434700B; CN106434700A; CN109825516A

Abstract

The invention provides a saccharomyces cerevisiae spt15 site-directed saturation gene mutation method for improving ethanol yield. The method comprises the steps of obtaining spt15 site-directed saturated mutant genes of saccharomyces cerevisiae by using a genetic engineering means, connecting the spt15 site-directed saturated mutant genes to an expression vector pYES2NTc, constructing a mutant library to obtain 12 single-point mutant genes in total, constructing recombinant plasmids with wild-type genes spt15, spt3 and a reference gene Neo gene, and transferring the recombinant plasmids into saccharomyces cerevisiae INVSC1 by using a lithium acetate method to obtain 15 recombinant saccharomyces cerevisiae strains. And (2) inoculating and fermenting the recombinant saccharomyces cerevisiae by taking glucose as a substrate, and determining the ethanol yield of the recombinant saccharomyces cerevisiae INVSC1-SPT15-M and INVSC1-SPT15-N, wherein the ethanol yield is greatly improved to 43.0 +/-0.9 g/L and 43.7 +/-0.2 g/L respectively. The increase of the strain is 17.8 percent and 19.7 percent compared with the control strain INVSC1-Neo respectively. The 2 mutant genes are respectively mutated from lysine to methionine and asparagine at position 127. The method is simple, the operation is easy, and the effect of improving the ethanol yield is obvious. Has good economic and social benefits.

Description

Saccharomyces cerevisiae site-directed saturation mutant gene spt15-N for improving ethanol yield and application thereof

The application is a divisional application named as a saccharomyces cerevisiae spt15 site-directed saturation gene mutation method for improving the ethanol yield, with the application number of 201610692224.2 and the application date of 20160819.

Technical Field

The invention relates to a technology for producing ethanol by biomass fermentation, in particular to a saccharomyces cerevisiae spt15 site-specific saturated gene mutation method for improving ethanol yield

Background

The traditional bioethanol production mainly takes grain fermentation as a main part, and the shortage phenomenon of grains in the world severely limits the source of raw materials. Lignocellulose is the most abundant biomass resource in the world, the quantity is the largest, the price is the lowest, and the total annual output accounts for about 50 percent of all biomass resources^[1]However, most of these substances are not used efficiently at present. Industrial ethanol prepared from biomass as a raw material is a clean renewable energy source, is a necessary trend to replace fossil fuels such as petroleum, and has been incorporated into strategic development plans of many countries.

Saccharomyces cerevisiae is a eukaryotic microorganism with high cell wall thickness, high sterol content, and high tolerance to toxic factors from ethanol and lignocellulosic hydrolysates [3 ]; the ethanol can be produced by rapidly fermenting glucose under the conditions of low pH and strict anaerobic condition, and byproducts are few; is not easy to be polluted by bacteria and virus, and the related industrial technology is mature. Saccharomyces cerevisiae whole genome sequencing has been completed [4], and is the best studied eukaryotic microorganism so far, and can be carried out by genetic manipulation and reconstruction of metabolic networks by using bioinformatics methods and means and increasingly sophisticated molecular biology techniques. Is a major and primary target microorganism for ethanol fermentation. Ethanol tolerance of yeast is related to multiple genes, so that the aim of improving ethanol tolerance and ethanol yield of yeast is difficult to achieve through traditional single gene knockout or overexpression. In 2006, Alper et al reported that the method of gTME improves ethanol yield and tolerance of yeast, and provides a new idea for yeast ethanol yield and tolerant metabolic engineering operation.

Transcriptional level regulation is one of the most efficient links in gene expression regulation. Global transcription mechanism engineering (gTME) is a technique for optimizing cell phenotype by gene transcription rearrangement^[6]It establishes initial transcription factor mutation library, modifies global transcription regulating factor, and carries out directional screening aiming at target product or target phenotype by molecular biology method, such as error-prone PCR, DNA shuffling, etc., so that the whole transcription regulating process is changed to change or improve the transcription and expression of target gene, thus obtaining the strain with enhanced target metabolic flow or enhanced specific phenotype. Transcription is performed by RNA polymerase, and in eukaryotes, RNA polymerase II is responsible for transcription to produce mRNA of most functional genes, and the transcription efficiency of RNA polymerase II is determined by the binding ability of initiation transcription factors and promoters. The spt15, one of the transcription initiation factors in Saccharomyces cerevisiae, is part of the transcription initiation complex and is a TATA binding protein^[7]And is involved in the formation of transcription initiation complex, controlling gene expression efficiency. The change of the combination ability of the TATA and a related gene promoter region influences the expression efficiency of the related gene, thereby causing the phenotypic change of strains. Its mutation makes the related gene over-expressed, and phenotypically raises the ethanol tolerance of yeast.

Chinese patent: a mutant saccharomyces cerevisiae initial transcription factor and its coding gene and application (ZL200810024036.8) it utilizes the random mutation technology to mutate the transcription factor spt15 gene, obtain a mutant spt15-6 of spt15 through screening; another chinese patent: the mutated saccharomyces cerevisiae initial transcription factor gene and the expression vector and application thereof (ZL201310008539.7) also use the random mutation technology to mutate the transcription factor spt15 gene, and obtain a mutant spt15-10 of spt15 through screening. There was a clear change in their alignment with the original s.cerevisiae spt15 gene sequence. The recombinant saccharomyces cerevisiae is constructed by utilizing a gene engineering technology, and the ethanol yield is changed by fermentation detection, but is not ideal for engineering application, namely the ethanol yield is further improved.

The invention obtains an initial transcription regulatory factor gene spt15 from a wild yeast strain through amplification, obtains a mutant gene by using a site-directed saturation mutation method, constructs a pYES2NTc vector, converts the vector into saccharomyces cerevisiae INVSC1 through a lithium acetate method for expression, and measures the yield of the obtained recombinant saccharomyces cerevisiae ethanol. 2 point mutation spt15 genes are obtained by screening. The metabolic characteristics of the recombined saccharomyces cerevisiae after mutation are greatly improved, namely the ethanol yield of the recombined saccharomyces cerevisiae is greatly improved.

Reference to the literature

[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 hong Mei, xu Lin, Yanming, etc. gTME constructs recombinant saccharomyces cerevisiae for co-fermenting xylose and glucose, the report of bioengineering, 2008,24(6): 16.

[2]Hal A,Gregory S.Global transcription machinery engineering:A newapproach for improving cellular phenotype.Metab Eng,2007,9:258 267.

Disclosure of Invention

The invention provides a method for improving ethanol yield by carrying out site-specific saturated gene mutation on spt15 of saccharomyces cerevisiae by using a genetic engineering means.

The present invention is thus achieved. A method for improving the site-specific saturation gene mutation of saccharomyces cerevisiae spt15 with ethanol yield comprises the following steps:

1. amplification of Saccharomyces cerevisiae INVSC1spt15 gene and recombinant plasmid construction

a) Extracting the genome of S.cerevisiae INVSC1 by using a genome extraction kit. The saccharomyces cerevisiae initial transcription factor Spt15 gene was amplified by PCR using Spt15_ Forward and Spt15_ Reverse primers with s. PCR cycling parameters were 94 ℃ for 5 min; 1min at 94 ℃, 1min at 56 ℃, 2min at 72 ℃ and 30 cycles. The amplified gene was purified and recovered by using a gel recovery kit (TaKaRa).

Wherein the primer: spt15_ Forward 5'-ATGGCCGATGAGGAACGTTTAAAGGAGTTTA-3'

Spt15_Reverse 5'-TCACATTTTTCTAAATTCACTTAGCACAGGGTATATAG-3'

The sequence of the original spt15 gene is shown in a sequence table SEQ ID No.1

b) The recovered spt15 gene is connected with pMD18-T (TaKaRa company) vector, the connecting vector is transformed into Escherichia coli DH5 alpha competent cells by calcium chloride method, monoclonal recombinant plasmid pMD18-spt15 is extracted, and the gene sequence spt15 is obtained by sequencing. The Spt15 gene on the pMD18-Spt15 plasmid was then PCR amplified using the Spt15_ yes2ntF and Spt15_ yes2ntR primers. PCR cycling parameters were 94 ℃ for 5 min; 1min at 94 ℃, 1min at 56 ℃, 2min at 72 ℃ and 30 cycles. The amplified gene was purified and recovered by a gel recovery kit (TaKaRa) after double digestion with Not I and BamH I.

Wherein the primer: spt15_ yes2ntF 5'-GGATCCGCCGATGAGGAACGTTTA-3'

Spt15_yes2ntR 5'-GCGGCCGCTCACATTTTTCTAAATTCAC-3'

c) The recovered spt15 gene is connected with a pYES2NTc linear vector which is subjected to NotI and BamHI double enzyme digestion and recovery, the connecting vector is transformed into an escherichia coli DH5 alpha competent cell by a calcium chloride method, a monoclonal recombinant plasmid pYES2NTc-spt15 is extracted, and the gene sequence spt15 is correct by sequencing.

2. Site-directed saturation mutant gene library and acquisition of recombinant saccharomyces cerevisiae

a) Through homologous alignment and three-dimensional structural analysis of the spt15 sequence, the Lys127 site is located at the middle binding site of the symmetric structure, and the mutation of the site can influence the binding of spt15 and spt3, so that the site is selected as a mutation site.

b) According to the principle of reverse overlap extension PCR (overlap extension PCR), a pair of mutation primers Spt15_ mutF and Spt15_ mutR are designed, wherein the lower-case-lined letter part is a mutation site and corresponds to a 127-th amino acid codon.

Firstly, using recombinant plasmid pYES2NTc-Spt15 as a template, using primers Spt15_ mutF and Spt15_ mutR to carry out plasmid amplification, wherein PCR amplification cycle parameters are 94 ℃ 50sec, 62 ℃ 45sec, 72 ℃ 7min, 30 cycles, finally, 72 ℃ extension 10min, reverse amplification is carried out to obtain a linear fragment containing a vector sequence and a gene sequence, the template is digested by Dpn I enzyme, glue is recovered,and (3) self-connection, transforming escherichia coli DH5 alpha, screening transformants by a kanamycin-resistant plate, and sequencing to determine whether the mutant gene is a mutant gene. Mutant gene sequence and original saccharomyces cerevisiae spt15 gene sequence^[10]The alignment showed that 12 different point mutations were obtained for lysine 127, and no mutations were found at other positions. The results of 12 point mutations are shown in Table 1.

Wherein the primer: spt15_ mutF 5' -ATGGTTGTTACCGGTGCAnnkAGTGAGGATGACTCA-3'

Spt15_mutR 5'-TGCACCGGTAACAACCATTTTCCCTGAGGCAAAAATTAAAGC-3'

c) The spt15 gene recombinant plasmids containing mutations and non-mutated were subjected to lithium acetate method^[9]Transformed into Saccharomyces cerevisiae INVSC 1. The SX screening culture medium is used for screening transformants to obtain a series of recombinant saccharomyces cerevisiae (total 12) containing the spt15 mutant gene, and the recombinant saccharomyces cerevisiae INVSC1-spt15-X is named as series recombinant saccharomyces cerevisiae according to the mutant gene number, wherein X is F, V, R, M, L, G, T, S, Q, D, N, I (see Table 1).

TABLE 1 site-directed saturation of mutant site codons and amino acids

3. And (3) carrying out fermentation experiment, result analysis and screening on the recombinant Saccharomyces cerevisiae INVSC1-spt 15-X.

Respectively inoculating the 12 recombinant saccharomyces cerevisiae and the reference component obtained in the previous step into 50mL of seed culture medium, culturing for 24h at 30 ℃ under 200r/min, inoculating 10% (V/V) into a 500mL triangular flask containing 100mL of fermentation culture medium, properly diluting the bacterial liquid, measuring the absorbance value at 600nm, and respectively inoculating the strains in the logarithmic phase into 100mL of fermentation culture medium for anaerobic fermentation culture at 30 ℃ under 200 r/min. And (5) measuring the ethanol content and the residual sugar content. The fermentation broth sample was filtered through a 0.45 μm acetate fiber membrane, and ethanol concentration and glucose concentration were measured using SBA-40C biosensor analyzer and reagents (institute of biological research, academy of sciences, Shandong province). Through determination analysis and screening, the recombinant bacteria recombinant Saccharomyces cerevisiae INVSC1-SPT15-M and INVSC1-SPT15-N have greatly improved ethanol yield by using glucose, and the ethanol yield in a glucose culture medium is 43.0 +/-0.9 g/L and 43.7 +/-0.2 g/L respectively. An increase of 17.8% and 19.7% over the control strain, respectively.

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

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

The principle of the present invention is as such. The phenotype of a cell is determined by a combination of genes, and the construction of a strain with a desired phenotype requires the simultaneous modification of multiple genes, however, the ability to introduce these modifications is often limited. Gene expression regulation occurs at various levels of genetic information transfer, and transcriptional regulation is one of the most important links in gene expression regulation. Global transcription machinery engineering methods allow for the alteration of the expression of many terminal genes. I.e., by altering the response to the transcribed protein, so that a large perturbation of the entire transcript occurs. By modifying the initial transcription regulatory factor spt15, the optimized target phenotype is obtained by using specific screening conditions, namely, the simultaneous change of multiple genes is realized by changing the initial transcription factor so as to regulate the whole metabolic network.

The invention selects Lys127 site as mutation point to carry out site-directed saturation mutation, and obtains obvious technical effect.

The invention has obvious advantages. The method is simple, the operation is easy, and the effect of improving the ethanol yield is obvious. Has good economic and social benefits.

Drawings

FIG. 1 shows the cloning of spt15 gene and the electrophoretic detection and validation of pYES2NTc-spt15 recombinant plasmid

Wherein (A) is the PCR product of 1: spt 15; m is DNA marker DL2000

(B)1, double enzyme digestion verification of pMD18-spt15 plasmid; m is DNA marker DL10000

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

3, pYES2NTc-spt15 single enzyme digestion product; m is DNA marker DL5000

FIG. 2 sugar utilization of mutant Gene recombinant Yeast INVSC1-spt15-X

FIG. 3 ethanol yield of mutant Gene recombinant Yeast INVSC1-spt15-X versus control Strain

FIG. 4 shows the ethanol production and glucose utilization curves of mutant recombinant yeast strains INVSC1-SPT15-M, INVSC1-SPT15-N and control recombinant strain INVSC 1-Neo;

FIG. 5 three-dimensional structure and mutation site of the initial transcription regulatory factor gene spt15 in wild-type yeast strain;

Detailed Description

The invention is further illustrated by the following examples:

example 1: cloning of spt15 Gene and construction of recombinant plasmid thereof

Genome DNA of S.cerevisiae INVSC1 is used as a template, a designed primer is used for carrying out PCR reaction, and an obvious band is detected in about 0.75kb by electrophoresis of a PCR product (figure 1). The PCR product is connected with a cloning vector pMD18-T after being purified and recovered, the connection product is transformed into Escherichia coli E.coli DH5 alpha, a positive transformant pMD18-spt15 is screened out by using blue white spots, and the plasmid sequencing result shows that the obtained spt15 sequence is correct and has 100 percent of homology with the gene sequence in a gene bank (GenBank gene number M29459.1, protein number AAA 34458.1).

PCR amplification was performed on plasmid pMD18-spt15, double digestion was performed with Not I and BamH I, and agarose gel electrophoresis was performed to recover and purify the DNA. The recovered spt15 gene is connected with a pYES2NTc linear vector which is subjected to NotI and BamHI double enzyme digestion and recovery, a recombinant plasmid pYES2NTc-spt15 is obtained through PCR and enzyme digestion verification, and a gene sequence spt15 is obtained through sequencing.

Example 2 acquisition of site-directed saturation mutant Gene library and recombinant Saccharomyces cerevisiae

Using the recombinant plasmid pYES2NTc-spt15 obtained in example 1 as a template, reverse overlap extension PCR was performed, digested with Dpn I enzyme, recovered, and self-ligated to transform E.coli DH 5. alpha. kanamycin-resistant platesAnd (5) sequencing the transformant to identify whether the transformant is a mutant gene. Mutant gene sequence and original saccharomyces cerevisiae spt15 gene sequence^[10]The alignment showed that 12 different point mutations were obtained for lysine 127, and no mutations were found at other positions. The results of 12 point mutations are shown in Table 1.

Example 3 mutant Gene recombinant Saccharomyces cerevisiae screening

The spt15 gene recombinant plasmids containing mutations and non-mutated were subjected to lithium acetate method^[9]Transformed into Saccharomyces cerevisiae INVSC 1. Screening transformants by using a screening medium lacking uracil to obtain a series of recombinant Saccharomyces cerevisiae containing the spt15 mutant gene. The mutant genes are respectively named as series recombinant Saccharomyces cerevisiae INVSC1-spt15-X according to the sequence number, wherein X is K, F, V, R, M, L, G, T, S, Q, D, N, I (see Table 1). Wherein K is unmutated.

Example 4 sugar utilization of the INVSC1-spt15-X mutant Gene recombinant Yeast

Most genes show a variable reduction in the rate of glucose consumption. In particular SPT15-N, SPT 15-K. However, the control strain and the mutant gene recombinant strain both consume glucose completely within 12 hours, which shows that the glucose consumption rate does not influence the ethanol yield. (see FIG. 3)

Example 5 detection of mutant Gene recombinant Yeast INVSC1-spt15-X ethanol production by anaerobic fermentation

The yields of ethanol obtained after fermentation of the control strain INVSC1-Neo and the recombinant strain INVSC1-spt15-X in 100g/L glucose medium at 30 ℃ for 48h at 200r/min are shown in Table 2.

Compared with a control bacterium INVSC1-Neo, the amounts of the ethanol produced by INVSC1-SPT15-L, INVSC1-SPT15-G, INVSC1-SPT15-K, INVSC1-SPT15-I and INVSC1-SPT15-S are not changed basically, and the highest peaks are all at 24 h. And the ethanol yield of the INVSC1-SPT15-T, the INVSC1-Spt3, the INVSC1-SPT15-Q, the INVSC1-SPT15-F, the INVSC1-SPT15-V and the INVSC1-SPT15-D is obviously reduced, and the ethanol yield is about 25-29 g/L. Is 68.5% -79.5% of the control strain. While the highest ethanol yield of SPT15-R did not vary much compared to the control, the time to reach the highest yield of ethanol was postponed from 24h to 48 h.

The recombinant yeasts INVSC1-SPT15-M and INVSC1-SPT15-N have greatly improved ethanol yield by utilizing glucose, and the ethanol yield in a glucose culture medium is 43.0 +/-0.9 g/L and 43.7 +/-0.2 g/L respectively. An increase of 17.8% and 19.7% over the control strain, respectively. The results preliminarily revealed that significant mutations in the gene of the initiation transcription factor spt15 significantly changed the metabolic pathways and metabolic flows of s.cerevisiae.

TABLE 2 comparison of ethanol production under the same conditions for recombinant and control strains

Description of the materials used in the present invention.

Escherichia coli DH5, Saccharomyces cerevisiae INVSC1 as a Saccharomyces cerevisiae host strain, and pYES2NTc as a yeast expression vector are all available.

The genomic DNA extraction kit used for the experiments was purchased from Shanghai Shunhua bioengineering Co., Ltd, restriction enzymes Not I and BamH I were purchased from NEB Co., pMD18-T vector and gel recovery kit were produced by Dalianbao bioengineering Co., Ltd (TaKaRa), primer synthesis, plasmid extraction kit, Taq Polymerase and dNTP mix ampicillin antibiotic were produced by Shanghai Bioengineering Co., Ltd, TransTaq DNA Polymerase High Fidelity PCR Polymerase was produced by holotype gold, and the determination of gene sequences was accomplished by Yingjun (Invitrogen) bioengineering Co., Ltd. Other reagents were analytically pure.

Culture medium

Escherichia coli was cultured in LB medium, to which 50. mu.g/mL of ampicillin was added. Saccharomyces cerevisiae was cultured in YPAD medium.

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

Screening medium (SX) (g/L) 6.7 of yeast nitrogen source (YNB) without amino acid, 1.3 of essential amino acid mixture (lacking uracil), 20 of glucose and 20 of agar powder.

The seed culture medium (g/L) comprises yeast powder 10, peptone 20 and glucose 20.

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

Sequence listing

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Claims

1. A saccharomyces cerevisiae site-directed saturation mutant gene spt15-N for improving ethanol yield is characterized in that the sequence of the spt15-N mutant gene is shown in SEQ ID No.3, and lysine is mutated into asparagine at 127 position of the mutant gene.

2. A recombinant expression vector of a saccharomyces cerevisiae site-directed saturation mutant gene spt15-N for improving the yield of ethanol is characterized in that the recombinant expression vector contains a spt15-N mutant gene shown in an order SEQ ID No. 3.

3. A recombinant saccharomyces cerevisiae of a saccharomyces cerevisiae site-directed saturation mutant gene SPT15-N for improving the yield of ethanol is characterized in that the recombinant saccharomyces cerevisiae contains a SPT15-N mutant gene shown as SEQ ID No.3 in sequence and is named as INVSC1-SPT 15-N.

4. The application of the recombinant saccharomyces cerevisiae of the saccharomyces cerevisiae site-directed saturation mutation gene spt15-N for improving the ethanol yield is characterized in that the recombinant saccharomyces cerevisiae of claim 3 is used for producing ethanol by fermentation by taking glucose as a substrate.