CN116948852B - Saccharomyces cerevisiae with low ethanol synthesis amount and high acetyl coenzyme A synthesis amount and application thereof - Google Patents
Saccharomyces cerevisiae with low ethanol synthesis amount and high acetyl coenzyme A synthesis amount and application thereof Download PDFInfo
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- CN116948852B CN116948852B CN202310891969.1A CN202310891969A CN116948852B CN 116948852 B CN116948852 B CN 116948852B CN 202310891969 A CN202310891969 A CN 202310891969A CN 116948852 B CN116948852 B CN 116948852B
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Classifications
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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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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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
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- Zoology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
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- Biotechnology (AREA)
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- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a saccharomyces cerevisiae with low ethanol synthesis amount and high acetyl coenzyme A synthesis amount and application thereof, belonging to the technical field of biology. The invention provides a recombinant saccharomyces cerevisiae strain, which aims to reduce the accumulation of main byproduct ethanol in saccharomyces cerevisiae, increase the synthesis amount of acetyl coenzyme A, modify MTH1, MED2 and HXT2 transcription factors, screen to obtain an optimal transcription factor combination for reducing the synthesis amount of ethanol, and compare heterogeneous acetyl coenzyme A synthesis routes of different synthesis routes integrated on saccharomyces cerevisiae genome. The strain can realize growth in a sterile culture medium with glucose as a carbon source, builds a platform for synthesizing high-value compounds by metabolic engineering modified saccharomyces cerevisiae, has a simple construction method, is convenient to use, and has good application prospect.
Description
Technical Field
The invention relates to saccharomyces cerevisiae with low ethanol synthesis amount and high acetyl coenzyme A synthesis amount and application thereof, belonging to the technical field of biology.
Background
Saccharomyces cerevisiae is also known as baker's yeast or budding yeast. Saccharomyces cerevisiae is the most widely related yeast to humans, and has been used in the brewing industry as a food-safe strain for making foods such as bread and steamed bread. In recent years, scholars have begun to research the use of Saccharomyces cerevisiae for the production of natural products such as arteannuic acid, notoginsenoside, etc. Saccharomyces cerevisiae has the advantages of high safety, low pathogenicity, high stress resistance, low probability of phage contamination and the like, so that the saccharomyces cerevisiae plays an important role in the field of genetic engineering. However, due to the strong ethanol synthesis capability, the yield of the products can be reduced when the products are synthesized, the production cost of the products can be increased, and meanwhile, the growth of strains can be inhibited by a large amount of ethanol accumulation, so that the synthesis of the products is not facilitated. acetyl-CoA is an important intermediate metabolite in Saccharomyces cerevisiae and can be used for the synthesis of terpenoids, lipids and amino acids. Cytosolic acetyl-CoA in Saccharomyces cerevisiae is mainly produced by acetic acid, which results in a decrease in the amount of acetyl-CoA synthesized when we inhibit ethanol synthesis. Therefore, the improvement of accumulation of acetyl-CoA has great potential for producing specific products by reducing ethanol synthesis through genetically engineering industrial production strains, and can provide an excellent production platform.
At present, the reduction of the ethanol synthesized by saccharomyces cerevisiae is mainly carried out by the following methods: by knocking out the pyruvate decarboxylase, the conversion of the pyruvate to the acetaldehyde is blocked, and the synthesis of the ethanol is reduced; the global transcription factor is changed to relieve the Crabtree effect, so that the synthesis of ethanol is reduced. By knocking out genes related to ethanol synthesis and increasing enzyme systems related to conversion of acetaldehyde to acetic acid and conversion of acetic acid to acetyl-CoA. However, the growth of the Saccharomyces cerevisiae strain for reducing ethanol synthesis is inhibited, and the synthesis amount of acetyl-CoA is insufficient, so that the application of the recombinant strain is limited.
Disclosure of Invention
In order to solve the problems, the invention provides a saccharomyces cerevisiae strain capable of reducing the synthesis amount of ethanol and improving the synthesis amount of acetyl coenzyme A, adopts the Cre/loxp technology to modify transcription factors, optimizes global metabolic pathway, reduces the synthesis of ethanol in the saccharomyces cerevisiae strain, simultaneously expresses heterogeneous acetyl coenzyme A synthesis pathway, can realize low-ethanol and high-acetyl coenzyme A production, has simple construction method, is convenient to use and has good application prospect.
A first object of the present invention is to provide a low ethanol synthetic Saccharomyces cerevisiae expressing
Transcription factor mutant MTH1 A81D&I85S; or (b)
Transcription factor mutants MTH1 A81D、MED2*432Y and HXT2 W466*; or (b)
Transcription factor mutants MTH1 I85S、MED2*432Y and HXT2 W466*; or (b)
Transcription factor mutants MTH1 A81D&I85S、MED2*432Y and HXT2 W466*;
Wherein, MTH1 A81D is obtained by mutating the 81 st alanine of the amino acid sequence shown in SEQ ID NO.18 into aspartic acid, MTH1 I85S is obtained by mutating the 85 th isoleucine of the amino acid sequence shown in SEQ ID NO.18 into serine, MTH1 A81D&I85S is obtained by mutating the 81 st alanine of the amino acid sequence shown in SEQ ID NO.18 into aspartic acid and the 85 th isoleucine into serine, MED2 *432Y is obtained by replacing the 432 th stop codon of the amino acid sequence shown in SEQ ID NO.19 with tyrosine, and HXT2 W466* is obtained by replacing the 466 rd tryptophan of the amino acid sequence shown in SEQ ID NO.20 with stop codon.
Further, the nucleotide sequences of the transcription factor mutants MTH1 A81D、MTH1I85S、MTH1A81D&I85S、MED2*432Y、HXT2W466* are shown as SEQ ID NO.1-5 respectively.
Further, saccharomyces cerevisiae CEN PK2-1C MATA is used; ura3-52; trp1-289; leu2-3,112; his3- Δ1; MAL2-8C; SUC2 is the host bacterium.
Further, the transcription factor mutant MTH1 A81D、MTH1I85S or MTH1 A81D&I85S was integrated into the MTH1 site, the transcription factor mutant MED2 *432Y was integrated into the MED2 site, and the transcription factor mutant HXT2 W466* was integrated into the HXT2 site.
Further, the Cre/loxp method is adopted to integrate the transcription factor mutant into the saccharomyces cerevisiae genome, and the specific construction method comprises the following steps:
1) Selecting defective amino acid tags and constructed transcription factor mutant plasmids, designing PCR primers to enable the overlapping area of adjacent segments of a gene expression frame to reach 40-100 bp, and constructing an MTH1 A81D,MTH1I85S,MTH1A81D&I85S,MED2*432Y,HXT2W466* transcription factor mutant expression integration frame;
2) The transcription factor mutant expression integration boxes are respectively integrated into the MTH1, MED2 and HXT2 sites of the saccharomyces cerevisiae by a Cre/loxp method.
The second object of the present invention is to provide a recombinant Saccharomyces cerevisiae having a low ethanol synthesis amount and a high acetyl-CoA synthesis amount, which is produced by using the above Saccharomyces cerevisiae having a low ethanol synthesis amount as a starting material and is heterologously expressed
A pyruvate oxidase-encoding gene po and a phosphotransacetylase-encoding gene pta; or (b)
Phosphoenolpyruvate carboxylase encoding gene ppc, malate thiokinase large subunit encoding gene mtkA, malate thiokinase small subunit encoding gene mtkB, malate coa lyase encoding gene mcl, and hydroxypyruvate reductase encoding gene hprA.
Further, the nucleotide sequence of gene po is shown as SEQ ID NO.10, the nucleotide sequence of gene pta is shown as SEQ ID NO.12, the nucleotide sequence of gene ppc is shown as SEQ ID NO.13, the nucleotide sequence of gene mtkA is shown as SEQ ID NO.14, the nucleotide sequence of gene mtkB is shown as SEQ ID NO.15, the nucleotide sequence of gene mcl is shown as SEQ ID NO.16, and the nucleotide sequence of gene hprA is shown as SEQ ID NO. 17.
Further, gene po and gene pta are integrated at position 1021; gene ppc, gene mtkA, gene mtkB, gene mcl and gene hprA were integrated into the PCK1 site.
Further, gene po is expressed by promoter P TDH3, gene pta by promoter P GPD, gene ppc by promoter P TEF1, gene mtkA by promoter P GPD, gene mtkB by promoter P TDH3, gene mcl by promoter P ADH1, gene hprA by promoter P SED1.
Further, gene po is expressed by terminator T ATP5, gene pta by terminator T CYC1, gene ppc by terminator T ADH1, gene mtkA by terminator T CYC1, gene mtkB by terminator T ATP5, gene mcl by terminator T TDH3, gene hprA by terminator T PGK1.
Further, the heterologous acetyl coenzyme A anaplerotic pathway is integrated into the saccharomyces cerevisiae genome by adopting a Cre/loxp technology, and the specific construction method comprises the following steps:
1) Selecting defective amino acid tag, heterologous acetyl coenzyme A anaplerotic pathway gene, constitutive promoter P TEF1,PGPD,PTDH3,PADH1 or P SED1, terminator T ADH1,TCYC1,TATP5,TTDH3 or T PGK1, designing PCR primer to make overlap region of adjacent segment of gene expression frame reach 40-100 bp, constructing gene expression integration frame of PO-PTA or PPC-MtkA-MtkB-McL-HPRA.
2) The acetyl-CoA expression integration box is integrated into the Saccharomyces cerevisiae 1021 or PCK1 gene locus by the Cre/loxp method, respectively.
The third object of the present invention is to provide the use of the above-mentioned low ethanol synthetic amount Saccharomyces cerevisiae or low ethanol synthetic amount, high acetyl-CoA synthetic amount recombinant Saccharomyces cerevisiae in biosynthesis.
Further, the reduction of the synthesis amount of the ethanol is realized in the biosynthesis, the growth inhibition of the ethanol accumulation on the strain is reduced, the synthesis of related products is promoted, the carbon conversion rate and the biomass yield of the saccharomyces cerevisiae are improved, and the capability of synthesizing high-value compounds by the saccharomyces cerevisiae is enhanced.
A fourth object of the present invention is to provide a method for producing acetyl-CoA or its metabolite, comprising the step of fermentative production using the above-mentioned low ethanol synthesis amount Saccharomyces cerevisiae or recombinant Saccharomyces cerevisiae.
Further, acetyl-CoA metabolites include, but are not limited to, fatty acids, ketone bodies, cholesterol, 3-hydroxypropionic acid, squalene, and the like.
Further, glucose is used as a substrate for fermentation production.
Further, the recombinant strain is cultured for 16h to 24h at 28 ℃ to 32 ℃ and 180 rpm to 260rpm to obtain seed liquid, the seed liquid is transferred into a fermentation culture medium at an inoculation rate of 2 percent to 5 percent, and the seed liquid is aerated and fermented at the pH=6.0 to 8.0 and 180 rpm to 260rpm and 28 ℃ to 32 ℃.
The invention provides a transcription factor mutant MTH1 A81D&I85S, which is obtained by mutating the 81 st alanine of an amino acid sequence shown in SEQ ID NO.18 into aspartic acid and mutating the 85 th isoleucine into serine.
The invention also provides a gene for encoding the transcription factor mutant MTH1 A81D&I85S, a recombinant plasmid or a host cell containing the transcription factor mutant MTH1 A81D&I85S, and the host cell can select Saccharomyces cerevisiae, so that the constructed recombinant Saccharomyces cerevisiae not only can remarkably reduce the synthesis amount of ethanol, but also can promote the growth of host bacteria, and has great potential in biosynthesis.
The invention also claims the application of the transcription factor mutant MTH1 A81D&I85S in reducing the ethanol synthesis amount of the strain.
The invention has the beneficial effects that:
The Cre/loxp method is adopted to reform MTH1, MED2 and HXT2 transcription factors, different transcription factors are mutated or combined, the optimal transcription factor combination for reducing the synthesis amount of ethanol is obtained through screening, and the recombinant strain is constructed to greatly reduce the accumulation of ethanol in the fermentation production process, so that the growth inhibition of the strain is favorably relieved. In addition, the invention obtains the saccharomyces cerevisiae strain with low ethanol synthesis capability and high acetyl-CoA accumulation capability through comparing heterogeneous acetyl-CoA synthesis paths of different synthesis paths integrated on the saccharomyces cerevisiae genome and finally through integrating and expressing the optimal combination of transcription factors and the acetyl-CoA synthesis paths.
Drawings
FIG. 1 shows the ethanol production of the transcription factor mutant recombinant strain.
FIG. 2 shows the growth of recombinant strains of transcription factor mutants.
FIG. 3 shows the acetyl-CoA yields of recombinant strains of different heterologous acetyl-CoA synthetic pathways.
FIG. 4 is the yield of ethanol in recombinant strains.
FIG. 5 shows fatty acid production in recombinant strains.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The materials and methods involved in the following examples are as follows:
(1) Sequence information:
The nucleotide sequences of the transcription factor mutant MTH1 A81D,MTH1I85S,MTH1A81D&I85S,MED2*432Y,HXT2W466* are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
The transcription factor mutant is subjected to gene integration expression through a Cre/loxp system as shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8. The transcription factor mutant expression cassette was integrated into the Saccharomyces cerevisiae MTH1, MED2 and HXT2 sites.
The relevant gene sequences of the acetyl-coa anaplerotic pathway are: XPK SEQ ID, PO, SEQ ID, 10, XSFPK SEQ ID, 11, PTA, 12, PPC, 13, MTKA SEQ ID, 14, mtkB SEQ ID, 15, mcL SEQ ID, 16, HPRA SEQ ID, 17.
The amino acid sequence of the MTH1 wild type is shown as SEQ ID NO. 18. MTH1 A81D refers to the mutation of alanine at position 81 of the sequence shown in SEQ ID No.18 to aspartic acid, MTH1 I85S refers to the mutation of isoleucine at position 85 to serine, MTH1 A81D&I85S refers to the mutation of alanine at position 81 to aspartic acid and isoleucine at position 85 to serine.
The amino acid sequence of the MED2 wild type is shown as SEQ ID NO. 19. MED2 *432Y refers to the replacement of the 432 th stop codon of the sequence shown in SEQ ID No.19 with tyrosine.
The amino acid sequence of HXT2 wild type is shown in SEQ ID NO. 20. HXT2 W466* refers to replacing tryptophan at position 466 of the sequence shown in SEQ ID NO.20 with a stop codon.
(2) Method of
All recombinant s.cerevisiae strains in the present invention were fermented by: recombinant s.cerevisiae recombinant strains were streaked on amino nitrogen source-free plates (lacking the corresponding amino acids of the defect) and incubated at 30℃until a large number of colonies were grown.
Fatty acid gas chromatography-mass spectrometer (GC-MS) detection method: shimadzu (GCMS-QP 2010 SE), SH-Rtx-Wax chromatographic column, mobile phase of He, column temperature of 100 ℃, sample injection amount of 1 mu L, split ratio of 20:1 and flow of 1.0ml/min.
Ethanol high performance liquid chromatography detection method: agilent (1260 Infinicity II), organic acid chromatographic column, mobile phase 5mM H 2SO4, column temperature 55 ℃, differential detector temperature 40 ℃, sample injection amount 10 μl, and flow rate 0.6ml/min.
High performance liquid chromatography detection method of acetyl coenzyme A: ZORBAX Eclipse Plus C18 chromatography column (250 m. Times.4.6 mm. Times.5 μm); 90% of sodium phosphate buffer (A) with a mobile phase of 0.2mol/L and 10% of acetonitrile (B); the flow rate is 1mL/min; column temperature 25 ℃; ultraviolet detection wavelength 254nm; the sample injection amount was 10. Mu.L.
EXAMPLE 1 construction of recombinant Saccharomyces cerevisiae Strain with Low ethanol Synthesis
This example constructs a mutant MTH1 A81D,MTH1I85S,MTH1A81D&I85S,MED2*432Y,HXT2W466* transcription factor plasmid. According to the design method of the overlapped derivative PCR primer, the primer is designed to enable the overlapped area of the adjacent fragments of the gene expression frame to reach 40-100 bp. CEN PK2-1C MATA in S.cerevisiae; ura3-52; trp1-289; leu2-3,112; his3- Δ1; MAL2-8C; SUC2 as strain 1. Primers are designed to amplify the upstream and downstream homology arms of the gene integration frames on both sides of the MTH1 locus of the Saccharomyces cerevisiae CEN PK2-1C chromosome. The mutant transcription factor MTH1 A81D plasmid is used as a template, and a primer is designed to amplify the mutant transcription factor. PCR was performed using plasmid pMHyLp-LEU as a template (shown as SEQ ID NO. 6), and the defective expression cassette fragment was obtained by amplification, and the gene integration cassette was obtained by overlap extension PCR. And transferring the obtained gene expression frame into saccharomyces cerevisiae to obtain the strain 2. Strains 3 and 4 were obtained by engineering MTH1 I85S and MTH1 A81D&I85S, respectively, in the same procedure. Primers are designed to amplify the upstream and downstream homology arms of the gene integration frames on both sides of the MED2 locus of the Saccharomyces cerevisiae CEN PK2-1C chromosome. The mutant MED2 *432Y plasmid of the transcription factor is used as a template, and a primer is designed to amplify the mutant transcription factor. PCR was performed using plasmid pMHyLp-HIS as a template (shown as SEQ ID NO. 7), and the defective expression cassette fragment was obtained by amplification, and the gene integration cassette was obtained by overlap extension PCR. The obtained gene expression cassette is transferred into saccharomyces cerevisiae to obtain strain 5. Primers were designed to amplify the upstream and downstream homology arms of the gene integration frames on both sides of the HXT2 site of the Saccharomyces cerevisiae CEN PK2-1C chromosome. The mutant HXT2 W466* plasmid of the transcription factor is used as a template, and a primer is designed to amplify the mutant transcription factor. PCR was performed using plasmid pMHyLp-TRP as a template (shown as SEQ ID NO. 8), and the defective expression cassette fragment was obtained by amplification, and the gene integration cassette was obtained by overlap extension PCR. The obtained gene expression cassette is transferred into saccharomyces cerevisiae to obtain strain 6. Strain 7 was obtained by combining transcription factor mutants MTH1 A81D and HXT2 W466*. Strain 8 was obtained by combining transcription factor mutants MTH1 I85S and HXT2 W466*. Strain 9 was obtained by combining transcription factor mutants MTH1 A81D&I85S and HXT2 W466*. Strain 10 was obtained by combining the transcription factor mutants MED2 *432Y and HXT2 W466*. Strain 11 was obtained by combining transcription factor mutants MTH1 A81D,MED2*432Y and HXT2 W466*. Strain 12 was obtained by combining the transcription factor mutants MTH1 I85S,MED2*432Y and HXT2 W466*. Strain 13 was obtained by combining the transcription factor mutants MTH1 A81D&I85S,MED2*432Y and HXT2 W466*.
The results of measuring the ethanol yield synthesized by fermenting strains 1 to 13 for 96 hours (see FIG. 1) show that the variation trend of the ethanol yield in the constructed strain of 13 transcription factor mutants is not consistent, but the ethanol yield is reduced under the combination of double mutation and triple mutation, especially about 50% under the combination of triple mutation. The constructed gene integration frame is transformed into saccharomyces cerevisiae competent cells through a lithium acetate transformation method, colonies are selected for PCR verification, and partial PCR correct transformants are selected for sequencing verification.
The growth of recombinant strains introduced with different transcription factor mutants was also measured, and the results are shown in FIG. 2. It can be seen that the MTH1 mutant significantly promoted the growth of the strain, but the growth of the MED2 and HXT2 and the strain under the combined conditions was not significantly altered compared to the wild strain.
EXAMPLE 2 construction of high-AcetylCoA recombinant Saccharomyces cerevisiae Strain and detection of acetyl-CoA Synthesis
The XPK and PTA genes were codon optimized according to Saccharomyces cerevisiae codon bias and total gene synthesis was performed (gene sequences shown as SEQ ID NO.9 and SEQ ID NO.12, respectively). According to the design method of the overlapped derivative PCR primer, the primer is designed to enable the overlapped area of the adjacent fragments of the gene expression frame to reach 40-100 bp. Primers were designed to amplify the upstream and downstream homology arms of the gene integration cassettes on both sides of the 1021 locus of the Saccharomyces cerevisiae CEN PK2-1C chromosome, promoters P TEF1,PGPD,PTDH3,PADH1 and P SED1, and terminators T ADH1,TCYC1,TATP5,TTDH3 and T PGK1. The full-gene synthesized XPK and PTA plasmid are used as templates, and primers are designed to amplify XPK and PTA genes. PCR was performed using plasmid pMHyLp-LEU as a template (shown as SEQ ID NO. 6), and the defective expression cassette fragment was obtained by amplification, and the gene integration cassette was obtained by overlap extension PCR. And transferring the obtained gene expression frame into saccharomyces cerevisiae to obtain the strain A-1. The same PO and PTA (gene sequences are shown as SEQ ID NO.10 and SEQ ID NO. 12) plasmids synthesized by the whole genes are used as templates, XFSPK and PTA (gene sequences are shown as SEQ ID NO.11 and SEQ ID NO. 12) plasmids are used as templates, and A-2-A-3 strains are respectively constructed according to the method. The PPC, mtkA, mtkB, mcL and HPRA genes were codon optimized according to the Saccharomyces cerevisiae codon preference and subjected to total gene synthesis (the gene sequences are shown in SEQ ID NO.13-17, respectively). According to the design method of the overlapped derivative PCR primer, the primer is designed to enable the overlapped area of the adjacent fragments of the gene expression frame to reach 40-100 bp. Primers were designed to amplify the upstream and downstream homology arms of the PCK1 locus on both sides of the Saccharomyces cerevisiae CEN PK2-1C chromosome, promoters P TEF1,PGPD,PTDH3,PADH1 and P SED1, and terminators T ADH1,TCYC1,TATP5,TTDH3 and T PGK1. The primer is designed to amplify PPC, mtkA, mtkB, mcL and HPRA genes by taking PPC, mtkA, mtkB, mcL and HPRA plasmids synthesized by the whole genes as templates. PCR was performed using plasmid pMHyLp-LEU as a template (shown as SEQ ID NO. 6), and the defective expression cassette fragment was obtained by amplification, and the gene integration cassette was obtained by overlap extension PCR. And transferring the obtained gene expression frame into saccharomyces cerevisiae to obtain the strain A-4. The constructed gene integration frame is transformed into saccharomyces cerevisiae competent cells through a lithium acetate transformation method, colonies are selected for PCR verification, and partial PCR correct transformants are selected for sequencing verification.
The acetyl-CoA content in the different recombinant strains was examined as follows: taking 10m L fermentation liquor, centrifuging at 8,000 r/min and 4 ℃ for 10min,0.25mol/L, washing twice by 1mL of phosphate buffer (phosphate buffer saline, PBS), centrifuging at 8,000 r/min and 4 ℃ for 10min, removing supernatant, re-suspending by 1mL of 6% perchloric acid, dropwise adding 0.3mol/L potassium carbonate solution for salt precipitation (fully and uniformly mixing in the adding process), adjusting the pH value to 3.0,12,000 r/min, and centrifuging at 4 ℃ for 10min to remove potassium perchlorate (KClO 4) crystals.
The results are shown in FIG. 3, in which the content of acetyl-CoA in the recombinant strains A-2 and A-4 is significantly increased in the logarithmic growth phase compared with the wild strain.
EXAMPLE 3 construction of Low ethanol Synthesis of high acetyl-CoA Synthesis Strain
The optimal acetyl-CoA synthesis pathway was expressed on the basis of the recombinant strains (11, 12 and 13) having the lowest ethanol synthesis amount in example 1. According to the design method of the overlapped derivative PCR primer, the primer is designed to enable the overlapped area of the adjacent fragments of the gene expression frame to reach 40-100 bp. Primers were designed to amplify the upstream and downstream homology arms of the PCK1 locus on both sides of the Saccharomyces cerevisiae CEN PK2-1C chromosome, promoters P TEF1,PGPD,PTDH3,PADH1 and P SED1, and terminators T ADH1,TCYC1,TATP5,TTDH3 and T PGK1. The primer is designed to amplify PPC, mtkA, mtkB, mcL and HPRA genes by taking PPC, mtkA, mtkB, mcL and HPRA plasmids synthesized by the whole genes as templates. PCR was performed using plasmid pMHyLp-LEU as a template (shown as SEQ ID NO. 6), and the defective expression cassette fragment was obtained by amplification, and the gene integration cassette was obtained by overlap extension PCR. And transferring the obtained gene expression frame into 11,12 and 13 bacteria to obtain strains 14-16. The constructed gene integration frame is transformed into saccharomyces cerevisiae competent cells through a lithium acetate transformation method, colonies are selected for PCR verification, and partial PCR correct transformants are selected for sequencing verification.
EXAMPLE 4 recombinant Saccharomyces cerevisiae fermentation
Single colony is selected to a seed culture medium (YPD sterile culture medium taking glucose as a carbon source is selected as the seed culture medium), and the culture is carried out for 16-24 hours at 30 ℃ and 220rpm until the cell growth logarithmic phase.
Seed culture solution is inoculated into fermentation culture medium (YPD sterile culture medium using glucose as carbon source is selected as fermentation culture medium) according to initial inoculation amount of 1-3%, and is cultured for 72h at 30 ℃ at 220 rpm. After 72h, the culture was stopped, the supernatant of the fermentation broth was taken and the yeast cells after fermentation were centrifuged and lyophilized for subsequent detection and analysis.
Example 5 detection of ethanol Synthesis and fatty acid Synthesis by recombinant Saccharomyces cerevisiae Strain
The ethanol and fatty acids in all recombinant s.cerevisiae strains of the present invention were extracted in the following manner. Taking fermentation broth after fermentation, centrifuging at 12000rpm for 5min, sucking supernatant with 1ml syringe, passing the fermentation broth supernatant through 0.22 μm water-based filter membrane for removing impurities, and detecting by liquid chromatography.
Pentadecanoic acid was added as an internal standard to the lyophilized cells, methanol was added: the chloroform and glass beads were broken by shaking, and then the broken solution was transferred to a new volumetric flask, again 1.5ml of methanol/chloroform solvent was added for extraction, the procedure was repeated twice, and the resulting lipid extracts were combined, and 1ml of chloroform and 3ml of aqueous solution were added to the combined solution. The sample was vigorously shaken, centrifuged at 10000rpm for 5min, the upper liquid was discarded, the lower organic phase was transferred to a new glass tube, after which 1ml sulfuric acid was added: methanol is heated in a water bath kettle at 95 ℃ for 90min, taken out and cooled, n-hexane and NaCl aqueous solution are added, supernatant is removed by centrifugation, and then the determination can be carried out by using GC-MS (the sum of the contents of C16:0, C16:1, C18:0 and C18:1 is used for the fatty acid content change).
The ethanol yield and fatty acid yield after fermentation of the recombinant strain are shown in figures 4-5. Compared with the original strain, the ethanol yield of the strain 14-16 is reduced by more than 66%, and the fatty acid yield is improved by about 75%.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (11)
1. A saccharomyces cerevisiae with low ethanol synthesis amount is characterized in that: the saccharomyces cerevisiae with low ethanol synthesis amount is prepared from saccharomyces cerevisiae S. CEREVISIAE CEN PK2-1C MATA, ura3-52, trp1-289, leu2-3,112, his 3-delta 1, MAL2-8C, SUC2 serving as host bacteria, and the transcription factors MTH1, MED2 and HXT2 on the host bacteria are mutated by Cre/loxp technology, and the mutated transcription factors are MTH1 I85S、MED2*432Y and HXT2 W466*;
Wherein MTH1 I85S is obtained by mutating isoleucine at position 85 of the amino acid sequence shown in SEQ ID NO.18 into serine, MED2 *432Y is obtained by replacing the stop codon at position 432 of the amino acid sequence shown in SEQ ID NO.19 with tyrosine, and HXT2 W466* is obtained by replacing tryptophan at position 466 of the amino acid sequence shown in SEQ ID NO.20 with stop codon.
2. The low ethanol synthetic saccharomyces cerevisiae according to claim 1, wherein: the nucleotide sequences of the transcription factors MTH1 I85S、MED2*432Y、HXT2W466* are respectively shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 5.
3. The low ethanol synthetic saccharomyces cerevisiae according to claim 1, wherein: the nucleotide sequence encoding the transcription factor MTH1 I85S was integrated into the MTH1 site, the nucleotide sequence encoding the transcription factor MED2 *432Y was integrated into the MED2 site, and the nucleotide sequence encoding the transcription factor HXT2 W466* was integrated into the HXT2 site.
4. A recombinant saccharomyces cerevisiae with low ethanol synthesis and high acetyl-CoA synthesis, which is characterized in that: heterologous expression of phosphoenolpyruvate carboxylase encoding gene ppc, malate thiokinase large subunit encoding gene mtkA, malate thiokinase small subunit encoding gene mtkB, malate coa lyase encoding gene mcl and hydroxypyruvate reductase encoding gene hprA using the low ethanol synthesis amount saccharomyces cerevisiae as a starting strain according to any one of claims 1-3.
5. The recombinant s.cerevisiae according to claim 4, wherein the recombinant s.cerevisiae is characterized in that it comprises: gene ppc, gene mtkA, gene mtkB, gene mcl and gene hprA were integrated into the PCK1 site.
6. The recombinant s.cerevisiae according to claim 4, wherein the recombinant s.cerevisiae is characterized in that it comprises: gene ppc is expressed by promoter P TEF1, gene mtkA by promoter P GPD, gene mtkB by promoter P TDH3, gene mcl by promoter P ADH1, and gene hprA by promoter P SED1.
7. The recombinant s.cerevisiae according to claim 4, wherein the recombinant s.cerevisiae is characterized in that it comprises: gene ppc is expressed by terminator T ADH1, gene mtkA by terminator T CYC1, gene mtkB by terminator T ATP5, gene mcl by terminator T TDH3, and gene hprA by terminator T PGK1.
8. Use of a low ethanol synthesis saccharomyces cerevisiae according to any of claims 1-3 for reducing ethanol synthesis.
9. Use of the recombinant s.cerevisiae according to any of claims 4-7 for acetyl-coa synthesis.
10. A method for producing acetyl-coa or a metabolite thereof, comprising the step of fermentation production using the recombinant saccharomyces cerevisiae according to any one of claims 4-7 with glucose as a substrate, wherein the acetyl-coa metabolite is a fatty acid or 3-hydroxypropionic acid.
11. The method according to claim 10, wherein: culturing the strain at 28-32deg.C and 180-260 rpm to obtain seed solution, transferring into fermentation medium, and fermenting at pH=6.0-8.0 and 180-260 rpm and 28-32deg.C under aeration.
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| CN117887601A (en) | 2024-04-16 |
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