CN114941001B - Construction method and application of metabolic engineering strain of Saccharomyces cerevisiae producing sakura chlorophyll - Google Patents

Abstract

The present invention realizes the de novo synthesis of sakurain by Saccharomyces cerevisiae using glucose as a carbon source for the first time through a multi-level metabolic engineering strategy. First, a complete synthetic pathway for producing sakurain starting from glucose is successfully constructed in Saccharomyces cerevisiae, and then the supply of sakurain precursors p-coumaric acid and malonyl-CoA is strengthened to increase the yield. The fermentation of sakurain by the metabolically engineered strain obtained in the present invention is the highest level of microbial production of sakurain reported so far.

Description

Construction method and application of metabolic engineering strain of Saccharomyces cerevisiae producing sakura chlorophyll

Technical Field

The invention belongs to the technical field of bioengineering, and relates to the construction and application of a metabolic engineering strain of saccharomyces cerevisiae producing sakura chlorophyll.

Background technique

Sakuranetin, also known as Sakura-tei, has a chemical name of 5,7-dihydroxy-4'-benzyloxyflavanone. It is a dihydroflavonoid compound originally isolated from the bark of cherry trees and is a plant phytoalexin. Current studies have found that sakuranetin has anti-inflammatory, anti-tumor and immunomodulatory effects, has a therapeutic effect on asthma, and has a wide range of medical application potential. At the same time, because of its high antioxidant activity, it can effectively fight against melanin deposition, improve dull skin tone, and play a role in whitening and rejuvenating the skin. It is highly favored in the cosmetics market, and its market size is constantly expanding.

At present, plant extraction is the main method for producing sakura glycosides. Although there are many plants that can produce sakura glycosides, the content of sakura glycosides that can be extracted from most of these plants is not high. In addition, due to the limited cost and technology of extracting sakura glycosides, mass production of sakura glycosides still faces many difficulties. Using synthetic biology to construct a microbial cell factory to produce sakura glycosides can overcome the limitations of upstream raw material supply, season, and site, and realizing green biomanufacturing is the most promising production direction. So far, there has been no report on the synthesis of sakura glycosides in yeast. The yeast engineering bacteria that produce sakura glycosides constructed by the present invention have a good prospect for promotion and application.

Summary of the invention

Based on the defects of various current methods, the purpose of the present invention is to construct a metabolic engineering strain of Saccharomyces cerevisiae with multi-enzyme co-expression, and apply it to the production of sakura cin, thus realizing the production of sakura cin in Saccharomyces cerevisiae for the first time.

The technical solution adopted in the present invention is as follows:

The method for constructing a sakura-producing engineered strain of Saccharomyces cerevisiae comprises the following steps:

1) Strains and plasmids: Escherichia coli DH5α was used for the construction and propagation of all plasmids; Saccharomyces cerevisiae CEN.PK2-1C was used as the starting strain;

2) Acquisition of relevant 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, synthetic fragments or available plasmids were used for PCR amplification; At4CL1 from Arabidopsis thaliana was amplified by using Arabidopsis thaliana cDNA as a template; the three codon-optimized genes AtPAL2, AtC4H and AtATR2 were all from Arabidopsis thaliana and obtained from plasmids pCfB2584 and pCfB2767, and CoNOMT was amplified by using rice cDNA as a template Amplification was performed; two expression cassettes including CYB5 from Saccharomyces cerevisiae and codon-optimized At4CL2 from Arabidopsis thaliana were directly amplified from pCfB2767 and pCfB2584, respectively; EcaroL amplified genomic DNA from Escherichia coli, 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 auxiliary plasmids pH1, pH2, pH3, pH4, pH5, pH6 or pUC19 to obtain gene expression cassette plasmids using restriction ligation or Gibson assembly;

3) Strain construction, including the following sub-steps:

3.1) Methods used for strain construction:

The CRISPR/Cas9 system was used to perform gene deletion and site-specific integration of DNA fragments in Saccharomyces cerevisiae strains using Cas9 and gRNA expression plasmids; to facilitate genetic manipulation, the Cas9 expression cassette was amplified from p42H-spCas9 and integrated into the IX-1 genomic locus in Saccharomyces cerevisiae CEN.PK2-1C, and the resulting strain C00:CEN.PK2-1C,IX1::TEFp-SpCas9-ADH2t was used as a host for DNA integration and biosynthetic pathway engineering; equimolar amounts of purified linearized fragments were then co-transformed into S.cerevisiae with the corresponding gRNA plasmids using the LiAc/ssDNA/PEG yeast transformation method, and transformants were selected on YPD plates supplemented with 200 μg/L of G418; clones were verified by colony PCR using Green Taq Mix; subsequently, these clones with correct module integration were cultured 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 sakuramin from glucose:

Based on the C00 strain, the pathway from phenylalanine to coumaric acid CIA was constructed in Saccharomyces cerevisiae by introducing phenylalanine lyase AtPAL2 and cinnamate hydroxylase AtC4H, and the strain C01 was marked; subsequently, P450 reductase AtATR2 was introduced, and the natural cytochrome CYB5 of Saccharomyces cerevisiae was overexpressed to construct the biosynthetic pathway from coumaric acid CIA to p-coumaric acid p-HCA, and the strain C02 was obtained; based on the C02 strain, tyrosine ammonia lyase TAL, 4-coumaric acid-CoA ligase 4CL, chalcone synthase CHS, chalcone isomerase CHI, and naringenin-7-O-methyltransferase NOMT were further introduced to construct a complete synthetic pathway for the de novo production of sakura chlorophyll from glucose, and the initial sakura chlorophyll-producing strain YHS02 was obtained;

3.3) Adjust the copy number of sakurabin synthesis gene to enhance sakurabin biosynthesis:

Based on the YHS02 strain, multiple copies of the phenylalanine ammonia lyase gene PAL, cinnamate hydroxylase C4H, tyrosine ammonia lyase TAL, chalcone synthase CHS and naringenin-7-O-methyltransferase NOMT in the sakura biosynthesis pathway were generated, and the galactose genes GAL1/7/10 were knocked out to obtain the strain YHS07;

3.4) Strengthening the supply of sakuramin precursor to coumaric acid by removing the rate-limiting factor in the aromatic amino acid synthesis pathway:

Based on the YHS07 strain, two DAHP synthase mutants ARO3 ^K222L and ARO4 ^K229L that are not inhibited by aromatic amino acids and a chorismate mutase mutant ARO7 ^G141S were selected to enhance the synthesis of tyrosine and phenylalanine, resulting in the engineered strain YHS09.

3.5) Optimizing L-phenylalanine branching and enhancing metabolic flux for sakuramin synthesis:

The metabolic flow of sakuramin synthesis was enhanced by knocking out the bypass genes pdc5 and aro10 in the tyrosine and phenylalanine synthesis pathways based on the YHS09 strain, and the strain YHS11 was obtained; then, the heterologous shikimate kinase EcaroL, endogenous phenolic acid dehydratase PHA2, chorismate synthase ARO2 and pentafunctional aromatic protein ARO1 from Escherichia coli were systematically overexpressed on the basis of the YHS11 engineered bacteria to successfully construct the strain YHS16:

3.6) Enhance the content of malonyl-CoA, the precursor of sakura phytochrome:

Optimizing the supply of precursors in the shikimic acid pathway to increase CGA production: Based on the YHS16 strain, the synthesis of another precursor of sakuramotoxin, malonyl-CoA, was enhanced by knocking out YPL062W and inserting the acetyl-CoA carboxylase ACC1 mutants ACC1 ^{S659A, S1157A} to obtain the engineered strain YHS18.

The present invention also adopts such a technical solution: the aforementioned engineering strain YHS18 is used in the production of sakura glycosides by using a shake flask fermentation method or a bioreactor fermentation method.

The present invention realizes the de novo synthesis of sakurain by Saccharomyces cerevisiae using glucose as a carbon source for the first time through a multi-level metabolic engineering strategy. Firstly, a complete synthetic pathway (PAL, TAL, C4H, ATR2, 4CL-1, CHS, CHI and NOMT) for producing sakurain from glucose was successfully constructed in Saccharomyces cerevisiae. The shake flask fermentation yield of the constructed metabolic engineering strain YHS02 was 9 mg/L. Then, we used three modular strategies to improve the yield of sakurain: (1) The first modular strategy was to enhance the supply of sakurain precursor (p-coumaric acid), including overexpression of endogenous genes ARO4 ^K229L , ARO3 ^K222L , ARO7 ^G141S , ARO1, ARO2 and PHA2, and overexpression of EcaroL from Escherichia coli. In addition, we introduced exogenous HaTal and MtPDH1 to enhance the synthesis of p-coumaric acid from tyrosine; (2) The second modular strategy was to knock out the bypass metabolic flow genes aro10 and pdc5 for aromatic amino acid synthesis; (3) The third modular strategy was to introduce acetyl-CoA carboxylase ACC1 mutants (ACC1 ^{S659A, S1157A} ) to enhance the synthesis of malonyl-CoA, another precursor of sakurain. The metabolically engineered strain YHS-18 obtained by the present invention produces 48.62 mg/L of sakura polyphenols in a shake flask fermentation in a YPD medium for 72 hours, and the maximum sakura polyphenols production in a 1L bioreactor-scaled fermentation tank fermentation is 158.84 mg/L, which is the highest level of sakura polyphenols produced by microbial methods reported so far.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the metabolic process of de novo synthesis of sakuramin by Saccharomyces cerevisiae.

FIG. 2 is a diagram showing the effects of different genes on the ability of yeast to synthesize sakuramin.

FIG. 3 is a diagram showing the fermentation process of YHS-18 in a 1L bioreactor.

Detailed ways

See Figure 1. The symbols in the figure are as follows: Glucose, glucose; 6-PF, fructose-6-phosphate; 3-PG, glyceraldehyde-3-phosphate; E-4-P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; DAHP, 3-deoxy-δ-arabinoheptulose-7-phosphate; DHQ, 3-deoxyhydroquinic acid; DHS, 3-dehydroshikimic acid; SHIK, shikimic acid; S3P, shikimic acid-3-phosphate; EPSP, enolpyruvate phosphoshikimic acid; CHA, chorismic acid; PPA, Prephenate; PPY, phenylpyruvate; L-Phe, L-phenylalanine; CIA, coumaric acid; p-HCA, p-coumaric acid; PAA, phenylacetaldehyde; 4-HPP, 4-hydroxyphenylpyruvate; 4-HPAA, 4-hydroxyphenylacetaldehyde; L-Tyr, L-tyrosine; Coumaroyl-CoA, coumaroyl-CoA; Acetyl-CoA, acetyl-CoA; Pyruvate, pyruvate; Malonyl-CoA, malonyl-CoA; Naringenin chalcone, naringenin chalcone; Naringenin, naringenin; Sakuranetin, sakuranetin; 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, pentafunctional aromatic protein; PHA2, endogenous prephenate dehydratase; AtPAL2, phenylalanine lyase; AtC4H, cinnamate hydroxylase; AtATR, P450 reductase; CYB5, Saccharomyces cerevisiae yeast native cytochrome; ARO10/PDC5, phenylpyruvate decarboxylase; MtPDH1, tyrosine prephenate dehydrogenase; ARO8, aminotransferase; HaTAL, tyrosine aminolyase; At4CL, p-coumaryl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; CoNOMT, naringenin-7-O-methyltransferase; ACC1 ^{S659A, S1157A} , acetyl-CoA carboxylase.

In this example, the sakura-producing engineered strain of Saccharomyces cerevisiae was constructed as follows:

1) Strains and plasmids: Escherichia coli DH5α was used for the construction and propagation of all plasmids; Saccharomyces cerevisiae CEN.PK2-1C was used as the starting strain;

2) Acquisition of relevant 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, synthetic fragments or available plasmids were used for PCR amplification; At4CL1 from Arabidopsis thaliana was amplified by using Arabidopsis thaliana cDNA as a template; the three codon-optimized genes AtPAL2, AtC4H and AtATR2 were all from Arabidopsis thaliana and obtained from plasmids pCfB2584 and pCfB2767, and CoNOMT was amplified by using rice cDNA as a template. Two expression cassettes including CYB5 from Saccharomyces cerevisiae and codon-optimized At4CL2 from Arabidopsis thaliana were directly amplified from pCfB2767 and pCfB2584, respectively; EcaroL amplified genomic DNA from Escherichia coli, and the two codon-optimized genes CHS and CHI were amplified from the corresponding cloning vectors pCHS and pCHI, respectively. Then, these candidate genes, promoters or terminators were cloned into the auxiliary plasmids pH1, pH2, pH3, pH4, pH5, pH6 or pUC19 using restriction ligation or Gibson assembly to obtain the gene expression cassette plasmids.

3) Strain construction, including the following sub-steps:

3.1) Methods used for strain construction:

The CRISPR/Cas9 system was employed to perform gene deletion and site-specific integration of DNA fragments in Saccharomyces cerevisiae strains using Cas9 and gRNA expression plasmids; to facilitate genetic manipulation, the Cas9 expression cassette was amplified from p42H-spCas9 and integrated into the IX-1 genomic locus in Saccharomyces cerevisiae CEN.PK2-1C, and the resulting strain C00:CEN.PK2-1C,IX1::TEFp-SpCas9-ADH2t was used as a host for DNA integration and biosynthetic pathway engineering; equimolar amounts of purified linearized fragments were then co-transformed into S. cerevisiae with the corresponding gRNA plasmids 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 module integration were cultured 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 sakuramin from glucose:

Based on the C00 strain, the pathway from phenylalanine to coumaric acid CIA was constructed in Saccharomyces cerevisiae by introducing phenylalanine lyase AtPAL2 and cinnamate hydroxylase AtC4H, and strain C01 was labeled; subsequently, P450 reductase AtATR2 was introduced and the natural cytochrome CYB5 of Saccharomyces cerevisiae was overexpressed to construct the biosynthetic pathway from coumaric acid CIA to p-coumaric acid p-HCA, and strain C02 was obtained.

3.3) Based on the C02 strain, tyrosine ammonia lyase TAL, 4-coumarate-CoA ligase 4CL, chalcone synthase CHS, chalcone isomerase CHI, and naringenin-7-O-methyltransferase NOMT were further introduced to construct a complete synthetic pathway for the de novo production of sakurain from glucose, and the galactose genes GAL1/7/10 were knocked out to obtain the initial sakurain-producing strain YHS02. The sakurain production reached 9 mg/L after 72 h of YPD shake flask fermentation.

3.4) Adjust the copy number of sakurabin synthesis gene to enhance sakurabin biosynthesis:

Based on the YHS02 strain, multiple copies of the phenylalanine ammonia lyase gene PAL, tyrosine ammonia lyase TAL, chalcone synthase CHS and naringenin-7-O-methyltransferase NOMT in the sakura glycoside synthesis pathway were added to obtain the strain YHS07. The sakura glycoside production reached 13.37 mg/L after 72 hours of shake flask fermentation.

3.5) Strengthening the supply of sakuramin precursor to coumaric acid by removing the rate-limiting factor in the aromatic amino acid synthesis pathway:

Based on the YHS07 strain, we chose to overexpress two DAHP synthase mutants ARO3 ^K222L and ARO4 ^K229L that are not inhibited by aromatic amino acids and the chorismate mutase mutant ARO7 ^G141S to enhance the synthesis of tyrosine and phenylalanine. The resulting engineered strain YHSO9 had a yield of 16.51 mg/L in 72 hours of shake flask fermentation.

3.6) Optimizing L-phenylalanine branching and enhancing metabolic flux for sakuramin synthesis:

The metabolic flow of sakurain synthesis was enhanced by knocking out the bypass genes pdc5 and aro10 in the tyrosine and phenylalanine synthesis pathways based on the YHSO9 strain, and the strain YHS11 was obtained; then, the heterologous shikimate kinase (EcaroL), endogenous phenolic acid dehydratase (PHA2), chorismate synthase (ARO2) and pentafunctional aromatic protein (ARO1) from Escherichia coli were systematically overexpressed on the basis of the YHS11 engineered bacteria, and we introduced exogenous MtPDH1 to strengthen the pathway of synthesizing p-coumaric acid from tyrosine. The successful strain YHS16 was constructed, and the sakurain production reached 46.10 mg/L in shake flask fermentation for 72 hours.

3.7) Enhance the content of malonyl-CoA, the precursor of sakura phytochrome:

Optimizing the supply of precursors in the shikimic acid pathway to increase CGA production: Based on the YHS16 strain, the synthesis of malonyl-CoA, another precursor of sakura glycosides, was enhanced by knocking out YPL062W and inserting acetyl-CoA carboxylase ACC1 mutants ACC1 ^{S659A, S1157A} . The resulting engineered strain YHS18 produced 48.62 mg/L of sakura glycosides in a shake flask fermentation for 72 hours.

Comparison of a series of engineered strains of Saccharomyces cerevisiae producing sakura glycoside

The present invention is the first to construct a biosynthetic pathway for de novo synthesis of sakurain using glucose as a substrate in Saccharomyces cerevisiae. The metabolically engineered strain YHS18 finally obtained produced 48.62 mg/L of sakurain in shake flask fermentation for 72 h (Table 1, Figure 2). The sakurain production in 11 bioreactor scale-up tank fermentation can reach 158.84 mg/L, which is the highest level of microbial production of sakurain reported so far (Figure 3).

Table 1 Successfully constructed engineering strains of Saccharomyces cerevisiae

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.