CN114941001B - Construction method and application of metabolic engineering strain for producing sakura primordium by saccharomyces cerevisiae - Google Patents

Construction method and application of metabolic engineering strain for producing sakura primordium by saccharomyces cerevisiae Download PDF

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CN114941001B
CN114941001B CN202210473378.8A CN202210473378A CN114941001B CN 114941001 B CN114941001 B CN 114941001B CN 202210473378 A CN202210473378 A CN 202210473378A CN 114941001 B CN114941001 B CN 114941001B
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sakura
saccharomyces cerevisiae
yhs
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钟卫鸿
何炎
涂帅
肖锋
范文佳
侯正雨
黄子炎
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Zhejiang University of Technology ZJUT
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Abstract

The invention realizes that the saccharomyces cerevisiae takes glucose as a carbon source to synthesize the sakura de novo for the first time through a multi-level metabolic engineering strategy, firstly successfully constructs a complete synthesis path for producing the sakura de novo from glucose in the saccharomyces cerevisiae, and then strengthens the supply of sakura de precursor to coumaric acid and malonyl-CoA to provide the yield. The metabolic engineering strain obtained by the invention ferments the sakura extract, which is the highest level of sakura extract produced by the microorganism method reported so far.

Description

Construction method and application of metabolic engineering strain for producing sakura primordium by saccharomyces cerevisiae
Technical Field
The invention belongs to the technical field of bioengineering, and relates to construction and application of a saccharomyces cerevisiae metabolic engineering strain for producing sakura extract.
Background
Sakura extract (Sakuranetin), also known as sakura pavilion, is known as 5, 7-dihydroxy-4' -benzyloxy flavanone, originally a dihydroflavonoid compound isolated from the bark of sakura, and is a plant protection extract for plants. The research at present finds that the sakura extract has anti-inflammatory activity, anti-tumor and immunoregulation effects, has therapeutic effect on asthma and has wide medical application potential. Meanwhile, the anti-aging agent has higher anti-oxidation activity, can effectively resist melanin deposition, improve dark complexion, play a role in whitening and tendering skin, is favored in the cosmetic market, and has a continuously expanding market scale.
At present, plant extraction is the main method for producing sakura, and although many plants can produce sakura, the content of most of sakura which can be extracted by the plants is not high, and large-scale production of sakura still faces more difficulties due to the limited cost and technology for extracting sakura. The production of sakura extract by utilizing the plant of microorganism cells constructed by synthetic biology can overcome the limitations of upstream raw material supply, seasons and places, and the realization of green biological production is the most promising production direction. So far, no report exists on the synthesis of the sakura extract in the yeast, and the yeast engineering bacteria for producing the sakura extract constructed by the invention have good popularization and application prospects.
Disclosure of Invention
Based on the defects of various methods at present, the invention aims to construct a multienzyme co-expressed saccharomyces cerevisiae metabolic engineering bacterium, and apply the strain to the production of sakura extract, so as to realize the production of sakura extract in the saccharomyces cerevisiae for the first time.
The technical scheme adopted by the invention is as follows:
the construction method of the sakura production sakura engineering strain comprises the following steps:
1) Strains and plasmids: coli DH 5. Alpha. Was used for the construction and propagation of all plasmids; taking Saccharomyces cerevisiae CEN.PK2-1C as an initial strain;
2) Acquisition of related genes: all natural promoters, genes and terminators were amplified by PCR using Saccharomyces cerevisiae CEN.PK2-1C genomic DNA or available plasmids as templates; for codon optimized heterologous genes, PCR amplification is performed using synthetic fragments or available plasmids; at4CL1 from arabidopsis was amplified by using arabidopsis cDNA as a template; three genes AtPAL, atC H and AtATR2, codon optimized, were all from arabidopsis, obtained from plasmids pCfB2584 and pCfB2767, coNOMT amplified by using rice cDNA as template; the two expression cassettes included CYB5 from saccharomyces cerevisiae and the codon optimized At4CL2 from arabidopsis thaliana were amplified directly from pCfB2767 and pCfB2584, respectively; ecaroL amplification of genomic DNA from e.coli and the codon optimized two genes CHS and CHI from the corresponding cloning vectors pCHS and pCHI, respectively, then cloning these candidate genes, promoters or terminators into helper plasmids pH1, pH2, pH3, pH4, pH5, pH6 or pUC19 using restriction ligation or Gibson assembly to obtain gene expression cassette plasmids;
3) The strain construction comprises the following substeps:
3.1 Strain construction method:
gene deletion and DNA fragment site-specific integration in saccharomyces cerevisiae strains using Cas9 and gRNA expression plasmids using a CRISPR/Cas9 system; to facilitate gene manipulation, cas9 expression cassette was amplified from p42H-spCas9 and integrated into the IX-1 genomic site in saccharomyces cerevisiae cen.pk2-1C, resulting strain C00: CEN.PK2-1C, IX1: : TEFp-SpCas9-ADH2t is used as a host for DNA integration and biosynthetic pathway engineering; equimolar amounts of purified linearized fragments were then co-transformed with the corresponding gRNA plasmids to s.cerevisiae using the LiAc/ssDNA/PEG yeast transformation method, and transformants were selected on YPD plates supplemented with 200 μg/L G418; clones were verified by colony PCR using Green Taq Mix; subsequently, these clones with correct modular integration were grown overnight in YPD liquid medium and then streaked on antibiotic-free plates to loop out the gRNA vector;
3.2 Construction of the pathway for de novo synthesis of sakurin from glucose:
Constructing a path from phenylalanine to coumaric acid CIA in saccharomyces cerevisiae by introducing phenylalanine lyase AtPAL2 and cinnamic acid hydroxylase AtC H on the basis of the strain C00, and marking the strain C01; then respectively introducing P450 reductase AtATR and overexpressing natural cytochrome CYB5 of saccharomyces cerevisiae, and constructing a biosynthetic pathway from coumaric acid CIA to P-coumaric acid P-HCA to obtain a strain C02; continuously introducing tyrosine ammonia lyase TAL, 4-coumarate-CoA ligase 4CL, chalcone synthase CHS, chalcone isomerase CHI and naringenin-7-O-methyltransferase NOMT on the basis of a C02 strain, constructing a complete synthesis path for producing sakura from beginning to end from glucose, and obtaining a sakura element-producing initial strain YHS02;
3.3 Adjusting the copy number of the sakura gene to enhance sakura biosynthesis:
On the basis of YHS strain, the phenylalanine ammonia lyase gene PAL, cinnamic acid hydroxylase C4H, tyrosine ammonia lyase TAL, chalcone synthase CHS and naringenin-7-O-methyltransferase NOMT in the synthetic pathway of the multicopy sakura extract are utilized, and galactose genes GAL1/7/10 are knocked out to obtain a strain YHS07;
3.4 Enhancing the supply of coumaric acid by the sakurin precursor by removing the rate limiting factor in the aromatic amino acid synthesis pathway:
On the basis of YHS strain, selecting two DAHP synthase mutants ARO3 K222L、ARO4 K229L and chorismate mutase mutant ARO7 G141S which are not inhibited by aromatic amino acid to enhance the synthesis of tyrosine and phenylalanine, and obtaining engineering bacteria YHS09;
3.5 Optimizing L-phenylalanine branching and enhancing metabolic flow of sakurin synthesis):
Enhancing the metabolic flux of sakura hormone synthesis by knocking out the shunt genes pdc5 and aro10 during the tyrosine and phenylalanine synthesis pathway on the basis of YHS strain 09, to obtain strain YHS; then, a strain YHS which is successfully constructed by systematically overexpressing heterologous shikimate kinase EcaroL, endogenous phenol acid dehydratase PHA2, chorismate synthase ARO2 and five-function aromatic protein ARO1 from Escherichia coli on the basis of YHS engineering bacteria was obtained:
3.6 Enhancement of content of malonyl-coa, a sakurin precursor:
Optimizing shikimate pathway precursor supply increases CGA yield: on the basis of YHS strain, the synthesis of malonyl-coa, another precursor of sakura, was enhanced by knocking out YPL062W while inserting acetyl-coa carboxylase ACC1 mutant ACC1 S659A,S1157A, resulting in engineering strain YHS.
The invention also adopts the following technical scheme: the engineering strain YHS is applied to the production of the sakura extract by adopting a shake flask fermentation method or a bioreactor fermentation method.
The invention realizes that the saccharomyces cerevisiae takes glucose as a carbon source to synthesize the sakura extract from the head for the first time through a multilevel metabolic engineering strategy. Firstly, a complete synthesis path (PAL, TAL, C, H, ATR2, 4CL-1, CHS, CHI and NOMT) for producing sakura extract from glucose is successfully constructed in Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the shake flask fermentation yield of the metabolic engineering strain YHS02 is 9mg/L. The sakurin yield was then improved by three module strategies: (1) The first modular strategy, which intensified the supply of sakurin precursor (p-coumaric acid), included over-expression of the endogenous genes ARO4 K229L、ARO3K222L、ARO7G141S, ARO1, ARO2 and PHA2, and over-expression of EcaroL from escherichia coli, additionally we tried to introduce exogenous HaTal and MtPDH1 to intensify the pathway for the synthesis of p-coumaric acid from tyrosine; (2) A second module strategy, namely knocking out bypass metabolic flow genes aro10 and pdc5 synthesized by aromatic amino acids; (3) A third modular strategy, the acetyl-coa carboxylase ACC1 mutant (ACC 1 S659A,S1157A) was introduced to enhance the synthesis of malonyl-coa, another precursor of sakura. The metabolic engineering strain YHS-18 obtained by the invention is subjected to shake flask fermentation in YPD culture medium for 72 hours to produce 48.62mg/L of sakura extract, and the yield of the sakura extract obtained by the method is up to 158.84mg/L when the sakura extract is amplified in a 1L bioreactor, so that the maximum level of sakura extract produced by a microorganism method is reported so far.
Drawings
FIG. 1 is a schematic diagram of the metabolism of sakura de novo synthesis of sakura de-novo.
FIG. 2 is a graph showing the effect of different genes on the capacity of yeast to synthesize sakura.
FIG. 3 is a diagram showing the fermentation process of YHS-18 in a 1L bioreactor.
Detailed Description
See fig. 1. The figures are labeled as follows: glucose; 6-P-F, fructose-6-phosphate; 3-P-G, glyceraldehyde-3-phosphate; E-4-P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-delta-arabinoheptulose 7-phosphate; DHQ, 3-deoxyhydroquinic acid; DHS, 3-dehydroshikimic acid; SHIK shikimic acid; S3P, shikimic acid-3-phosphate; EPSP, enolpyruvylshikimic acid; CHA, chorismate; PPA, prephenic acid; PPY, phenylpyruvate; L-Phe, L-phenylalanine; CIA, coumaric acid; p-HCA, p-coumaric acid; PAA, phenylacetaldehyde; 4-HPP, 4-hydroxyphenylpyruvic acid; 4-HPAA, 4-hydroxyphenylacetaldehyde; L-Tyr, L-tyrosine; coumaroyl-CoA, coumaroyl-CoA; acetyl-CoA, acetyl-CoA; pyruvate, pyruvic acid; malonyl-CoA, malonyl-CoA; NARINGENIN CHALCONE naringenin chalcone; NARINGENIN naringenin; sakuranetin, sakura extract; ARO3 K222L, L-phenylalanine feedback insensitive DAHP synthase; ARO4 K229L, L-tyrosine feedback insensitive DAHP synthase; AROB, 3-dehydroquinine synthase; ecaroL shikimate kinase II; ARO2, chorismate synthase; ARO7 G141S, L-tyrosine feedback insensitive chorismate mutase; ARO1, five-function aromatic protein; PHA2, endogenous prephenate dehydratase; atPAL2, phenylalanine lyase; atC4H, cinnamic acid hydroxylase; atATR, P450 reductase; CYB5, saccharomyces cerevisiae natural cytochrome; ARO10/PDC5, phenylpyruvate decarboxylase; mtPDH1, tyrosine prephenate dehydrogenase; ARO8, aminotransferase; haTAL tyrosine ammonolysis enzyme; at4CL, coumaroyl-coa ligase; CHS, chalcone synthetases; CHI, chalcone isomerase; coNOMT naringenin-7-O-methyltransferase; ACC1 S659A,S1157A, acetyl-coa carboxylase.
In this example, the Saccharomyces cerevisiae sakulargenin-producing engineering strain is constructed as follows:
1) Strains and plasmids: coli DH 5. Alpha. Was used for the construction and propagation of all plasmids; taking Saccharomyces cerevisiae CEN.PK2-1C as an initial strain;
2) Acquisition of related genes: all natural promoters, genes and terminators were amplified by PCR using Saccharomyces cerevisiae CEN.PK2-1C genomic DNA or available plasmids as templates; for codon optimized heterologous genes, PCR amplification is performed using synthetic fragments or available plasmids; at4CL1 from arabidopsis was amplified by using arabidopsis cDNA as a template; three genes AtPAL, atC H and AtATR2, codon optimized, were all from arabidopsis, obtained from plasmids pCfB2584 and pCfB2767, coNOMT were amplified using rice cDNA as template. The two expression cassettes included CYB5 from saccharomyces cerevisiae and the codon optimized At4CL2 from arabidopsis thaliana were amplified directly from pCfB2767 and pCfB2584, respectively; ecaroL genomic DNA from e.coli was amplified and codon optimized two genes CHS and CHI were amplified from the corresponding cloning vectors pCHS and pCHI, respectively, and then these candidate genes, promoters or terminators were cloned into helper plasmids pH1, pH2, pH3, pH4, pH5, pH6 or pUC19 using restriction ligation or Gibson assembly to obtain gene expression cassette plasmids.
3) The strain construction comprises the following substeps:
3.1 Strain construction method:
Gene deletion and DNA fragment site-specific integration in saccharomyces cerevisiae strains using Cas9 and gRNA expression plasmids using a CRISPR/Cas9 system; to facilitate gene manipulation, cas9 expression cassette was amplified from p42H-spCas9 and integrated into the IX-1 genomic site in saccharomyces cerevisiae cen.pk2-1C, resulting strain C00: CEN.PK2-1C, IX1: : TEFp-SpCas9-ADH2t is used as a host for DNA integration and biosynthetic pathway engineering; equimolar amounts of purified linearized fragments were then co-transformed with the corresponding gRNA plasmids to s.cerevisiae using the LiAc/ssDNA/PEG yeast transformation method and transformants were selected on YPD plates supplemented with 200 μg/LG 418; clones were verified by colony PCR using Green Taq Mix; subsequently, these clones with correct modular integration were grown overnight in YPD liquid medium and then streaked on antibiotic-free plates to loop out the gRNA vector.
3.2 Construction of the pathway for de novo synthesis of sakurin from glucose:
Constructing a path from phenylalanine to coumaric acid CIA in saccharomyces cerevisiae by introducing phenylalanine lyase AtPAL2 and cinnamic acid hydroxylase AtC H on the basis of the strain C00, and marking the strain C01; and respectively introducing P450 reductase AtATR and overexpressing the natural cytochrome CYB5 of the saccharomyces cerevisiae, and constructing a biosynthetic pathway from coumaric acid CIA to P-coumaric acid P-HCA to obtain the strain C02.
3.3 Continuously introducing tyrosine ammonia lyase TAL, 4-coumarate-CoA ligase 4CL, chalcone synthase CHS, chalcone isomerase CHI and naringenin-7-O-methyltransferase NOMT on the basis of a C02 strain, constructing a complete synthesis path for producing sakura from beginning to end from glucose, knocking out galactose genes GAL1/7/10, obtaining an initial strain YHS for producing sakura, and fermenting for 72h by YPD shaking until the sakura yield reaches 9mg/L.
3.4 Adjusting the copy number of the sakura gene to enhance sakura biosynthesis:
On the basis of YHS strain, the phenylalanine ammonia lyase gene PAL, tyrosine ammonia lyase TAL, chalcone synthase CHS and naringenin-7-O-methyltransferase NOMT in the synthetic pathway of the sakura extract are copied to obtain a strain YHS07, and the yield of the sakura extract is 13.37mg/L after shaking flask fermentation for 72 hours.
3.5 Enhancing the supply of coumaric acid by the sakurin precursor by removing the rate limiting factor in the aromatic amino acid synthesis pathway:
On the basis of YHS strain, two DAHP synthase mutants ARO3 K222L、ARO4K229L and chorismate mutase mutant ARO7 G141S which are not inhibited by aromatic amino acid are selected to enhance the synthesis of tyrosine and phenylalanine, and the obtained engineering bacterium YHSO9 is subjected to shake flask fermentation for 72h, and the yield reaches 16.51mg/L.
3.6 Optimizing L-phenylalanine branching and enhancing metabolic flow of sakurin synthesis):
Enhancing the metabolic flux of sakura hormone synthesis by knocking out the shunt genes pdc5 and aro10 during the tyrosine and phenylalanine synthesis pathway on the basis of YHSO strain to obtain strain YHS; next, heterologous shikimate kinase (EcaroL), endogenous phenol acid dehydratase (PHA 2), chorismate synthase (ARO 2) and five-functional aromatic protein (ARO 1) from E.coli were systematically overexpressed on the basis of YHS engineering bacteria, and we introduced an exogenous MtPDH1 to enhance the pathway for the synthesis of p-coumaric acid from tyrosine. The strain YHS is successfully constructed, and the yield of the sakura extract reaches 46.10mg/L after shaking flask fermentation for 72 hours.
3.7 Enhancement of content of malonyl-coa, a sakurin precursor:
optimizing shikimate pathway precursor supply increases CGA yield: based on YHS strain, synthesis of malonyl-coa, another precursor of sakura, was enhanced by knocking out YPL062W while inserting acetyl-coa carboxylase ACC1 mutant ACC1 S659A,S1157A. The yield of the obtained engineering strain YHS is 48.62mg/L after shake flask fermentation for 72 hours.
Comparison of sakura series engineering strains producing sakura
The invention constructs a biosynthesis way for synthesizing sakura essence from the head by taking glucose as a substrate in saccharomyces cerevisiae for the first time, and finally obtains metabolic engineering strain YHS which is fermented for 72 hours in a shaking bottle to obtain the sakura essence with the yield of 48.62mg/L (table 1, figure 2), and the yield of the sakura essence can reach 158.84mg/L when the sakura essence is fermented in an 11 bioreactor for amplification and upper tank, thus being the highest level of the sakura essence produced by a microorganism method reported so far (figure 3).
TABLE 1 Saccharomyces cerevisiae engineering bacteria which have been successfully constructed

Claims (2)

1. The construction method of the sakura production sakura engineering strain comprises the following steps:
1) Strains and plasmids: coli DH 5. Alpha. Was used for the construction and propagation of all plasmids; taking Saccharomyces cerevisiae CEN.PK2-1C as an initial strain;
2) Acquisition of related genes: all natural promoters, genes and terminators were amplified by PCR using Saccharomyces cerevisiae CEN.PK2-1C genomic DNA or available plasmids as templates; for codon optimized heterologous genes, PCR amplification is performed using synthetic fragments or available plasmids; at4CL1 from arabidopsis was amplified by using arabidopsis cDNA as a template; three genes AtPAL, atC H and AtATR2, codon optimized, were all from arabidopsis, obtained from plasmids pCfB2584 and pCfB2767, coNOMT amplified by using rice cDNA as template; the two expression cassettes included CYB5 from saccharomyces cerevisiae and the codon optimized At4CL2 from arabidopsis thaliana were amplified directly from pCfB2767 and pCfB2584, respectively; ecaroL amplification of genomic DNA from e.coli and the codon optimized two genes CHS and CHI from the corresponding cloning vectors pCHS and pCHI, respectively, then cloning these candidate genes, promoters or terminators into helper plasmids pH1, pH2, pH3, pH4, pH5, pH6 or pUC19 using restriction ligation or Gibson assembly to obtain gene expression cassette plasmids;
3) The strain construction comprises the following substeps:
3.1 Strain construction method:
Gene deletion and DNA fragment site-specific integration in saccharomyces cerevisiae strains using Cas9 and gRNA expression plasmids using a CRISPR/Cas9 system; to facilitate gene manipulation, cas9 expression cassette was amplified from p42H-spCas9 and integrated into the IX-1 genomic site in saccharomyces cerevisiae cen.pk2-1C, resulting strain C00: PK2-1C, IX1: TEFp-SpCas9-ADH2t is used as a host for DNA integration and biosynthetic pathway engineering; equimolar amounts of purified linearized fragments were then co-transformed with the corresponding gRNA plasmids to s.cerevisiae using the LiAc/ssDNA/PEG yeast transformation method, and transformants were selected on YPD plates supplemented with 200 μg/L G418; clones were verified by colony PCR using Green Taq Mix; subsequently, these clones with correct modular integration were grown overnight in YPD liquid medium and then streaked on antibiotic-free plates to loop out the gRNA vector;
3.2 Construction of the pathway for de novo synthesis of sakurin from glucose:
Constructing a path from phenylalanine to coumaric acid CIA in saccharomyces cerevisiae by introducing phenylalanine lyase AtPAL2 and cinnamic acid hydroxylase AtC H on the basis of the strain C00, and marking the strain C01; then respectively introducing P450 reductase AtATR and overexpressing Saccharomyces cerevisiae natural cytochrome CYB5, and constructing a biosynthetic pathway from coumaric acid CIA to P-coumaric acid P-HCA to obtain a strain C02; continuously introducing tyrosine ammonia lyase TAL, 4-coumarate-CoA ligase 4CL, chalcone synthase CHS, chalcone isomerase CHI and naringenin-7-O-methyltransferase NOMT on the basis of a C02 strain, constructing a complete synthesis path for producing sakura from beginning to end from glucose, and obtaining a sakura element-producing initial strain YHS02;
3.3 Adjusting the copy number of the sakura gene to enhance sakura biosynthesis:
On the basis of YHS strain, the phenylalanine ammonia lyase gene PAL, cinnamic acid hydroxylase C4H, tyrosine ammonia lyase TAL, chalcone synthase CHS and naringenin-7-O-methyltransferase NOMT in the synthetic pathway of the multicopy sakura extract are utilized, and galactose genes GAL1/7/10 are knocked out to obtain a strain YHS07;
3.4 Enhancing the supply of coumaric acid by the sakurin precursor by removing the rate limiting factor in the aromatic amino acid synthesis pathway:
On the basis of YHS strain, selecting two DAHP synthase mutants ARO3 K222L、ARO4 K229L and chorismate mutase mutant ARO7 G141S which are not inhibited by aromatic amino acid to enhance the synthesis of tyrosine and phenylalanine, and obtaining engineering bacteria YHS09;
3.5 Optimizing L-phenylalanine branching and enhancing metabolic flow of sakurin synthesis):
Enhancing the metabolic flux of sakura hormone synthesis by knocking out the shunt genes pdc5 and aro10 during the tyrosine and phenylalanine synthesis pathway on the basis of YHS strain 09, to obtain strain YHS; then, a strain YHS which is successfully constructed by systematically over-expressing heterologous shikimate kinase EcaroL, endogenous phenol acid dehydratase PHA2, chorismate synthase ARO2 and five-function aromatic protein ARO1 from escherichia coli on the basis of YHS engineering bacteria;
3.6 Enhancement of content of malonyl-coa, a sakurin precursor:
Optimizing shikimate pathway precursor supply increases CGA yield: on the basis of YHS strain, the synthesis of malonyl-coa, another precursor of sakura, was enhanced by knocking out YPL062W while inserting acetyl-coa carboxylase ACC1 mutant ACC1 S659A,S1157A, resulting in engineering strain YHS.
2. The use of the engineered strain YHS obtained by the method of claim 1 in the production of sakura, characterized in that: adopts a shaking flask fermentation method or a bioreactor fermentation method.
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