Method for synthesizing chicoric acid metabolic engineering strain from glucose and tartaric acid by saccharomyces cerevisiae

Abstract

The invention discloses a method for synthesizing a metabolic engineering strain of chicoric acid from glucose and tartaric acid by saccharomyces cerevisiae. The method comprises the following steps: starting from a metabolic strain YS08 for producing chlorogenic acid; the method comprises the following steps: introducing hydroxyl cinnamoyl transferase AtHCT into saccharomyces cerevisiae; on the other hand, Echinacea hydroxyl cinnamoyl coenzyme A: tartaric acid hydroxyl cinnamoyl transferase EpHTT is introduced to convert caffeic acid coenzyme A and exogenous tartaric acid into caffeoyl tartaric acid, and the caffeoyl tartaric acid is a chlorogenic acid producing strain, so that two precursors for synthesizing chicoric acid are obtained; meanwhile, introducing echinacea purpurea chicoric acid synthase EpCAS to catalyze chlorogenic acid and caffeoyl tartaric acid to generate chicoric acid so as to obtain a chicoric acid producing strain CA01; the copy number of the three genes AtHCT-EpTT-EpCAS is increased to two, a metabolic strain CA02 is obtained, the yield of the chicoric acid can reach 12.5 mg / L, and the purpose that the saccharomyces cerevisiae uses glucose and tartaric acid to synthesize the chicoric acid is achieved for the first time.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for synthesizing a metabolic engineering strain of cerevisiae from glucose and tartaric acid. Background Art

[0002] Cichoric acid, molecular formula C 22 H 18 O 12 , molecular weight 474.37112, is a caffeic acid derivative belonging to the phenolic acid class. It is widely found in various plants and plays an important role in human health.

[0003] Chicoric acid is a rare and valuable functional food ingredient with no significant dose-dependency, no overdose side effects, no contraindications, and no drug interactions. Chicoric acid has pharmacological effects on regulating glucose and lipid metabolism and is widely used in pharmaceuticals, nutritional supplements, and health foods. It also has anti-inflammatory, antioxidant, and anti-aging properties, as well as in combating digestive system diseases.

[0004] Industrial production of chicoric acid primarily relies on plant extraction. Although many plants can produce chicoric acid, plant production is limited by resource and environmental constraints, as well as the plant's slow growth cycle, making large-scale production of chicoric acid difficult. To meet growing market demand, the use of synthetic biology and metabolic engineering to construct metabolically engineered microbial strains capable of heterologous biosynthesis offers an alternative sustainable production method. To date, there have been no reports of producing chicoric acid from glucose and tartaric acid in Saccharomyces cerevisiae. Summary of the Invention

[0005] Based on the defects of various current methods, the present invention proposes for the first time a method for Saccharomyces cerevisiae metabolic engineering strain to synthesize chicoric acid from glucose and tartaric acid, and constructs a Saccharomyces cerevisiae metabolic engineering strain with multi-enzyme co-expression, realizing the efficient production of chicoric acid.

[0006] The technical problem to be solved by this invention is to provide a recombinant bacterium capable of efficiently producing chicoric acid using inexpensive substrates. Furthermore, the invention addresses the technical problems of constructing and applying this strain. This invention, for the first time, achieves chicoric acid synthesis in Saccharomyces cerevisiae using glucose as a carbon source through a multi-level metabolic engineering strategy. This is achieved primarily through the following means: 1. Based on the chlorogenic acid-producing strain YS08, the exogenous genes hydroxycinnamoyltransferase (HCT), tartrate hydroxycinnamoyltransferase (EpHTT), and Echinacea purpurea chicoric acid synthase (EpCAS) are inserted to introduce an optimal chicoric acid synthesis pathway; 2. These three genes are driven by the strong promoters TPI1p, TEF1p, and PGKp, respectively; and 3. The copy numbers of hydroxycinnamoyltransferase (HCT), tartrate hydroxycinnamoyltransferase (EpHTT), and Echinacea purpurea chicoric acid synthase (EpCAS) in the chicoric acid synthesis pathway are increased. The resulting metabolically engineered Saccharomyces cerevisiae strain CA02 can produce 12.5 mg / L of chicoric acid in shake flask fermentation, marking the first reported microbial production of chicoric acid.

[0007] Specifically, the method for constructing the chicoric acid-producing engineered strain of Saccharomyces cerevisiae comprises the following steps:

[0008] 1) Selection of strains and plasmids: Escherichia coli DH5α was used for all plasmid construction and propagation; Saccharomyces cerevisiae CEN.PK2-1C was used as the starting strain;

[0009] 2) DNA manipulation: All native promoters, genes, and terminators were PCR amplified 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. EpHTT and EpCAS from Echinacea were amplified using Echinacea cDNA as templates. The codon-optimized gene AtHCT was from Arabidopsis thaliana. The expression cassette promoters were driven by the strong promoters pTPI, pTEF, and pPGK, respectively. These candidate genes, promoters, or terminators were then cloned into helper plasmids using restriction ligation or Gibson assembly to obtain gene expression cassette plasmids.

[0010] 3) Strain construction:

[0011] 3.1) The CRISPR / Cas9 system was used to perform gene deletion and site-specific integration of DNA fragments in Saccharomyces cerevisiae 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. The resulting strain YT00:CEN.PK2-1C, IX1::TEFp-SpCas9-ADH2t was used as a host for DNA integration and biosynthetic pathway engineering. Equimolar amounts of the purified linearized fragments were then co-transformed with the corresponding gRNA plasmids into the Saccharomyces cerevisiae strain using the LiAc / ssDNA / PEG yeast transformation method, and transformants were selected on YPD plates supplemented with 200 μg / LG418. Clones were verified by colony PCR using GreenTaqMix. Subsequently, the correct clones were inoculated into YPD liquid medium, cultured overnight, serially passaged three times, and then plated on antibiotic-free plates to remove the gRNA vector.

[0012] 3.2) Construction of a de novo p-coumaric acid synthesis pathway from glucose using the phenylalanine pathway: A pathway from phenylalanine to coumaric acid (CIA) was constructed in Saccharomyces cerevisiae by introducing the phenylalanine lyase AtPAL2 and the cinnamate hydroxylase AtC4H into the YT00 strain, resulting in strain YT01. Subsequently, the P450 reductase AtATR2 was introduced, and the native yeast cytochrome CYB5 was overexpressed to construct the biosynthetic pathway from CIA to p-coumaric acid (p-HCA), resulting in strain YT02.

[0013] 3.3) Construction of a de novo chlorogenic acid pathway from glucose: Strain YC01 was generated by integrating quinate dehydrogenase EcYdiB, hydroxycinnamoyl-CoA quinoyltransferase CsHQT2, p-coumarate 3'-hydroxylase AtC3'H, cytochrome P450 reductase AtATR2, and 4-coumarate-CoA ligase 1At4CL1 into strain YT02;

[0014] 3.4) Increasing chlorogenic acid production by releasing carbon flux in the shikimate pathway: Overexpression of ARO4 in the YC01 strain K229L Mutants that maximize carbon flux into the SA pathway and increase CGA production; strains YC02, YC01, △trp1::TEF1p-ARO4 were obtained K229L -CYC1t;

[0015] 3.5) Optimizing L-phenylalanine branching and balancing the flux of p-HCA and QA to improve CGA yield: overexpressing Aro7 in the YC02 strain G141S, construct strain YC05, namely YC02, △ho-1::(HXT7t-ARO7 G141S -PGK1p)-(TPI1p-ARO3 K222L -TPI1t); then the single PHA2 overexpression strain YC0701 was constructed, namely YC05,III1::GPM1p-PHA2-ADH1t;

[0016] 3.6) Optimizing the supply of shikimate pathway precursors to increase CGA production: Based on the YC0701 strain, CRISPR-guided PYK1 D147N In vivo directed mutagenesis resulted in strain YC0702, namely YC0701, PYK11::PYK1 D147N ; After further overexpression of TKL1, strain YC0703, namely YC0702, XII5::PGK1p-TKL1-HXT7t, was obtained;

[0017] 3.7) Optimizing CGA biosynthesis by regenerating NADPH and adjusting the copy number of CGA synthesis genes: POS5 was overexpressed in strain YC0703 to promote intracellular NADPH production, thereby optimizing CGA biosynthesis, resulting in strain YC0704 (i.e., YC0703, Δho-2::GPDp-POS5); CGA production was increased by integrating the CGA biosynthesis pathway genes AtC3H and CsHQT2, resulting in strain YC0707 (i.e., YC0707, Δho-2::GPDp-POS5). 4, X2::ENO2p-HQT2-TPI1p-C3H; Based on YC0707, the strong endogenous promoter pPGK1 was inserted as a means of overexpressing the gene ZWF1 to construct the overexpression strain YS04, namely YC0707, ZWF1p::PGK1p; the GAL1p-RgTAL-CYCt expression cassette was integrated into the GAL80 locus of YS04 to generate YS08, namely YS04, ΔGAL80::GAL1p-RgTAL-CYC1t. YS08 is the starting strain for constructing a chicoric acid-producing strain;

[0018] 3.8) Using the pRS425-pTPI1-AtHCT plasmid as a template, PCR amplified the TPI1p-AtHCT-TPI1t-TEF1p expression cassette; the plasmid pRS425-TEF1p-AtF6'H1 was cut with BamHⅠ and XholⅠ restriction endonucleases to obtain the linearized pRS425 plasmid. Homologous sequences at both ends of the linearized vector were introduced at the 5' end of the forward and reverse amplification primers for tartrate hydroxycinnamoyltransferase EpHTT, so that the 5' and 3' ends of the amplified insert had and linear sequences, respectively. The two ends of the personalized cloning vector correspond to the same homologous sequence, and then the fragment after enzyme digestion is homologously recombined with tartrate hydroxycinnamoyltransferase EpHTT with the corresponding homology arm. The constructed plasmid is transformed into Escherichia coli DH5α to clone the plasmid to obtain plasmid pRS425-pTEF1-EpHTT. Using the pRS425-pTEF1-EpHTT plasmid as a template, PCR amplification of TEF1p-EpHTT-TEF1t expression cassette is performed; similarly, the plasmid pRS425-PG is cut with BamHI and HindIII restriction enzymes. K1p-At4CL1 was used to obtain the pRS425 linearized plasmid after enzyme digestion. The enzyme-digested fragment was homologously recombined with Echinacea purpurea chicoric acid synthase EpCAS with the corresponding homology arms. The constructed plasmid was transformed into Escherichia coli DH5α for cloning plasmid to obtain plasmid pRS425-pPGK1-EpCAS. The TEF1t-PGK1p-EpCAS-HXT7t expression cassette was amplified by PCR. Since the three expression cassettes each contained a 500 bp complementary sequence, equal amounts of linearized The fragment and gRNA were co-transformed into Saccharomyces cerevisiae cells, and the AtHCT gene from Arabidopsis thaliana and the EpHTT and EpCAS genes from Echinacea purpurea were introduced into the X4 locus of the YS08 strain using CRISPR / Cas9 technology to obtain strain CA01, namely YS08,X4::(TPI1p-AtHCT-TPI1t)-(TEF1p-EpHTT-TEF1t)-(PGK1p-EpCAS-HXT7t); the biosynthetic pathway of chicoric acid was constructed in strain CA01, and the synthesis of chicoric acid was achieved.

[0019] 3.9) Increasing chicoric acid production by increasing the copy number of the chicoric acid module: The copy number of each gene (hydroxycinnamoyltransferase HCT, Echinacea hydroxycinnamoyl-CoA:tartrate hydroxycinnamoyltransferase EpHTT, and Echinacea chicoric acid synthase EpCAS) was increased to two in strain CA01 to construct CA02, namely CA02,XVI::(TPI1p-AtHCT-TPI1t)-(TEF1p-EpHTT-TEF1t)-(PGK1p-EpCAS-HXT7t); strain CA02 successfully produced 12.5 mg / L of chicoric acid after 96 h of shake flask fermentation.

[0020] The present invention also provides an application method of the engineered bacteria CA02 in the fermentation production of chicoric acid as follows: the engineered strain CA02 is inoculated into a YPD culture medium, and cultured at 30°C and 220 rpm for 24 hours to obtain a seed liquid; an initial bacterial cell concentration OD660=0.05 is inoculated into a 250 mL Erlenmeyer flask containing 50 mL of seed culture medium, and 0.5 mL of 100 g / L tartaric acid solution (dissolved in pure methanol and filtered for sterilization) is added to the Erlenmeyer flask, and the fermentation is carried out at 30°C and 220 rpm, and samples are taken at intervals of 72 hours, 96 hours, and 120 hours to obtain a fermentation liquid of chicoric acid; the YPD culture medium composition: 20 g / L glucose, 10 g / L yeast powder, and 20 g / L peptone.

[0021] Compared with existing technologies, the present invention's main advantages are: It utilizes synthetic biology to engineer microbial cells to synthesize chicoric acid from glucose and tartaric acid, overcoming numerous challenges associated with plant-based extraction methods, such as raw material availability, seasonal restrictions, and environmental pollution, offering promising prospects for widespread application. The engineered strain CA02 was able to produce chicoric acid (CA) at a titer of 12.5 mg / L in shake flasks, a record high for microbial production to date. This also establishes a platform for the biosynthesis of chicoric acid and its derivative metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the metabolism of chicoric acid from glucose and tartaric acid in Saccharomyces cerevisiae;

[0023] Figure 2 This is a schematic diagram of the chicoric acid production of two strains CA01 and CA02 at different time periods;

[0024] Figure 3 This is the peak diagram of the product after 96h of shake flask fermentation of CA02 strain and verification by external standard method. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0026] Acquisition of relevant genes: Native promoters, terminators, and endogenous genes from Saccharomyces cerevisiae were amplified from CEN.PK2-1C genomic DNA. For codon-optimized heterologous genes, synthetic genes or available plasmids were used for PCR amplification. Hydroxycinnamoyltransferase (AtHCT) from Arabidopsis thaliana (its amino acid sequence is shown in SEQ ID NO. 1) was amplified using Arabidopsis thaliana cDNA as a template. Two codon-optimized genes, Echinacea purpurea hydroxycinnamoyl-CoA: tartrate hydroxycinnamoyltransferase (EpHTT, its amino acid sequence is shown in SEQ ID NO. 2) and Echinacea purpurea chicoric acid synthase (EpCAS, its amino acid sequence is shown in SEQ ID NO. 3), were synthesized by a biotechnology company. These candidate genes, promoters, or terminators were then cloned into helper plasmids using restriction ligation or Gibson assembly to generate gene expression cassette plasmids.

[0027] Example

[0028] like Figure 1 As shown in the figure: Glucose represents glucose; DAHP: 3-deoxy-δ-arabinoheptulose-7-phosphate; DHQ: 3-dehydroquinic acid; DHS: 3-dehydroshikimic acid; SA: shikimic acid; S3P: shikimic acid-3-phosphate; EPSP: 5-enolpyruvylshikimic acid-3-phosphate; CHA: chorismic acid; PPA: prephenylic acid; PPY: phenylpyruvic acid; L-Phe: L-phenylalanine; 4HPP: 4-hydroxyphenylpyruvic acid; L-Tyr: L-tyrosine; CIA: cinnamic acid; PA: p-coumaric acid; PA-CoA: p-coumaryl coenzyme A; QA: quinic acid; p-Coumaroyl quinate: p-coumarylquinic acid; CGA: chlorogenic acid; Caftaric acid acid: caffeoyltartaric acid; TA: tartaric acid; CA: chicoric acid; CFA: caffeic acid; CFA-CoA: caffeic acid coenzyme A; PYR: pyruvate. PYK1 D146N : Encoding pyruvate kinase 1 with reduced catalytic activity; ARO3 K222L : Encoding L-phenylalanine feedback-insensitive DAHP synthase; ARO4 K229L : Encoding L-tyrosine feedback-insensitive DAHP synthase; ARO7 G141S: encoding L-tyrosine feedback-insensitive chorismate mutase; PHA2: encoding prephenate dehydratase; AtPAL2: encoding phenylalanine ammonia lyase 2; AtC4H: encoding cinnamate hydroxylase; AtCPR1 / AtCPR2: encoding P450 reductase; CYB5: encoding yeast native pigment b5; At4CL1: encoding p-coumaroyl-CoA ligase; EcYdiB: encoding quinate dehydrogenase; CsHQT2: encoding quinate hydroxycinnamoyl-CoA transferase; AtC3'H: encoding p-coumarate hydroxylase; EpHTT: tartrate hydroxycinnamoyltransferase; AtHCT: hydroxycinnamoyltransferase; EpCAS: Echinacea cichoric acid synthase;

[0029] Method used for strain construction:

[0030] Saccharomyces cerevisiae CEN.PK2-1C (MATa; ura3-52; trp1-289; leu2-3112; his3Δ1; MAL2-8C; SUC2) was used as a host cell for chicoric acid production. Gene knockout and DNA insertion were performed using the CRISPR / Cas9 system. To facilitate genetic manipulation, a Cas9 expression cassette was amplified from p42H-spCas9 and integrated into the IX-1 genomic locus of Saccharomyces cerevisiae CEN.PK2-1C. The resulting strain, YT00:CEN.PK2-1C,IX1::TEFp-SpCas9-ADH2t, was used as a host for DNA integration and biosynthetic pathway engineering. Equal amounts of the linearized fragments and gRNA were co-transformed into Saccharomyces cerevisiae cells using the standard LiAc / ssDNA / PEG method. Strains were screened on YPD agar plates containing 200 μg / mL G418, and successful gene knockout or DNA insertion was verified by PCR amplification. Subsequently, the correct clones were inoculated into YPD liquid medium and cultured overnight, serially passaged three times, and then plated on antibiotic-free plates to remove the gRNA vector.

[0031] 1) The CRISPR / Cas9 system was used to perform gene deletion and site-specific integration of DNA fragments in Saccharomyces cerevisiae 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. The resulting strain YT00:CEN.PK2-1C, IX1::TEFp-SpCas9-ADH2t was used as a host for DNA integration and biosynthetic pathway engineering. Equimolar amounts of the purified linearized fragments were then co-transformed with the corresponding gRNA plasmids into the Saccharomyces cerevisiae strain 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 GreenTaqMix. Subsequently, correct clones were inoculated into YPD liquid medium, cultured overnight, serially passaged three times, and then plated on antibiotic-free plates to remove the gRNA vector.

[0032] 2) Construction of a de novo p-coumaric acid synthesis pathway from glucose based on the phenylalanine pathway: A pathway from phenylalanine to coumaric acid (CIA) was constructed in Saccharomyces cerevisiae by introducing the phenylalanine lyase AtPAL2 and the cinnamate hydroxylase AtC4H into the YT00 strain, resulting in strain YT01. Subsequently, the P450 reductase AtATR2 was introduced, and the native yeast cytochrome CYB5 was overexpressed to construct the biosynthetic pathway from CIA to p-coumaric acid (p-HCA), resulting in strain YT02.

[0033] 3) Construction of a de novo chlorogenic acid pathway from glucose: Strain YC01 was generated by integrating quinate dehydrogenase EcYdiB, hydroxycinnamoyl-CoA quinoyltransferase CsHQT2, p-coumarate 3'-hydroxylase AtC3'H, cytochrome P450 reductase AtATR2, and 4-coumarate-CoA ligase 1At4CL1 into strain YT02;

[0034] 4) Increasing chlorogenic acid production by releasing carbon flux in the shikimate pathway: overexpressing ARO4 in the YC01 strain K229L Mutants that maximize carbon flux into the SA pathway and increase CGA production; strains YC02, YC01, △trp1::TEF1p-ARO4 were obtained K229L -CYC1t;

[0035] 5) Optimizing L-phenylalanine branching and balancing the flux of p-HCA and QA to improve CGA yield: by overexpressing Aro7 in the YC02 strain G141S , construct strain YC05, namely YC02, △ho-1::(HXT7t-ARO7G141S -PGK1p)-(TPI1p-ARO3 K222L -TPI1t); then the single PHA2 overexpression strain YC0701 was constructed, namely YC05,III1::GPM1p-PHA2-ADH1t;

[0036] 6) Optimizing the supply of shikimate pathway precursors to increase CGA production: Based on the YC0701 strain, CRISPR was used to guide the expression of PYK1 D147N In vivo directed mutagenesis resulted in strain YC0702, namely YC0701, PYK11::PYK1 D147N ; After further overexpression of TKL1, strain YC0703, namely YC0702, XII5::PGK1p-TKL1-HXT7t, was obtained;

[0037] 7) Optimizing chlorogenic acid biosynthesis by regenerating NADPH and adjusting the copy number of CGA synthesis genes: POS5 was overexpressed in strain YC0703 to promote intracellular NADPH production, thereby optimizing CGA biosynthesis, resulting in strain YC0704, i.e., YC0703, Δho-2::GPDp-POS5; CGA production was increased by integrating the CGA biosynthesis pathway genes AtC3H and CsHQT2, resulting in strain YC0707, i.e., YC0704. ,X2::ENO2p-HQT2-TPI1p-C3H; based on YC0707, the strong endogenous promoter pPGK1 was inserted as a means of overexpressing the gene ZWF1 to construct the overexpression strain YS04, namely YC0707, ZWF1p::PGK1p; the GAL1p-RgTAL-CYCt expression cassette was integrated into the GAL80 locus of YS04 to obtain YS08, namely YS04, ΔGAL80::GAL1p-RgTAL-CYC1t. YS08 is the starting strain for constructing a chicoric acid-producing strain;

[0038] 8) Using the pRS425-pTPI1-AtHCT plasmid as a template, PCR amplified the TPI1p-AtHCT-TPI1t-TEF1p expression cassette; the plasmid pRS425-TEF1p-AtF6'H1 was cut with BamHⅠ and XholⅠ restriction endonucleases to obtain the linearized pRS425 plasmid. The homologous sequences at both ends of the linearized vector were introduced into the 5' end of the forward and reverse amplification primers for tartrate hydroxycinnamoyltransferase EpHTT, so that the 5' and 3' ends of the amplified insert had the same homologous sequences as the two ends of the linearized cloning vector. The homologous sequence should be consistent, and the fragment after enzyme digestion was homologously recombined with tartrate hydroxycinnamoyltransferase EpHTT with the corresponding homology arm. The constructed plasmid was transformed into Escherichia coli DH5α to clone the plasmid to obtain plasmid pRS425-pTEF1-EpHTT. Using pRS425-pTEF1-EpHTT plasmid as a template, TEF1p-EpHTT-TEF1t expression cassette was amplified by PCR. Similarly, plasmid pRS425-PGK1p-At4CL1 was digested by BamHⅠ and HindIII restriction endonucleases to obtain enzyme-digested pR The S425 linearized plasmid was used to homologously recombine the fragments after enzyme digestion with Echinacea chicoric acid synthase EpCAS with corresponding homology arms. The constructed plasmid was transformed into Escherichia coli DH5α to clone the plasmid to obtain the plasmid pRS425-pPGK1-EpCAS. The TEF1t-PGK1p-EpCAS-HXT7t expression cassette was amplified by PCR using the pRS425-pPGK1-EpCAS plasmid as a template. Since the three expression cassettes each contained a 500 bp complementary sequence, equal amounts of linearized plasmids were converted by LiAc / ssDNA / PEG transformation. The fragment and gRNA were co-transformed into Saccharomyces cerevisiae cells, and the AtHCT gene from Arabidopsis thaliana and the EpHTT and EpCAS genes from Echinacea purpurea were introduced into the X4 locus of the YS08 strain using CRISPR / Cas9 technology to obtain strain CA01, namely YS08,X4::(TPI1p-AtHCT-TPI1t)-(TEF1p-EpHTT-TEF1t)-(PGK1p-EpCAS-HXT7t). The biosynthetic pathway of chicoric acid was constructed in strain CA01, realizing the synthesis of chicoric acid from glucose and tartaric acid.

[0039] 9) Increasing chicoric acid production by increasing the copy number of the chicoric acid module: The copy number of the three genes of hydroxycinnamoyltransferase HCT, Echinacea hydroxycinnamoyl-CoA:tartrate hydroxycinnamoyltransferase EpHTT, and Echinacea chicoric acid synthase EpCAS in the CA01 strain was increased to two to construct CA02, namely CA01, XVI::(TPI1p-AtHCT-TPI1t)-(TEF1p-EPHTT-TEF1t)-(PGK1p-EpCAS-HXT7t);

[0040] The engineered bacteria constructed or used in the examples of the present invention are shown in Table 1, the constructed plasmids are shown in Table 2, and the primer sequences used are shown in Table 3.

[0041] Table 1 Successfully constructed engineered yeast strains of Saccharomyces cerevisiae

[0042]

[0043]

[0044] Table 2 Summary of constructed plasmids

[0045]

[0046]

[0047] Table 3 Summary of primers used

[0048]

[0049]

[0050]

[0051] The engineered strains CA01 and CA02 were inoculated into YPD medium and cultured at 30°C and 220rpm for 24h to obtain seed liquid; the initial bacterial concentration OD660 = 0.05 was inoculated into a 250mL triangular flask containing 50mL seed medium, and 0.5mL of 100g / L tartaric acid solution (dissolved in pure methanol and filtered and sterilized) was added to the triangular flask. The fermentation was carried out at 30°C and 220rpm, and samples were taken at intervals of 72h, 96h, and 120h to obtain the fermentation liquid of chicoric acid; YPD medium composition: 20g / L glucose, 10g / L yeast powder, 20g / L peptone. The yield of strains CA01 and CA02 at different time periods can be seen Figure 2 ,Depend on Figure 2 It was found that at 96h of shake flask fermentation, the maximum chicoric acid production of the CA02 strain reached 12.5mg / L.

[0052] The culture sample (600 μl) was mixed with an equal volume of methanol, shaken on a vortex shaker for 30 minutes, and centrifuged at 12,000 × g for 10 minutes. The supernatant was filtered and then processed by high-performance liquid chromatography. The supernatant was analyzed on an Agilent 1260HPLC instrument equipped with a reversed-phase C18 column (250 × 4.6 mm, 5 μm; Agilent, USA) and a UV detector. The instrument was operated at 40°C with a flow rate of 1 ml / min. The mobile phase was acetonitrile (B) and an aqueous solution containing 0.2% phosphoric acid (A). The following gradient was used: 5-35% solvent B for 15 minutes, 35-5% solvent B for 1 minute, and 5% solvent B for 5 minutes. The injection volume was 10 μL. As Figure 3 As shown, chicoric acid (CA) was detected at 330 nm (14.7 minutes), indicating the production of chicoric acid. The quantification of chicoric acid was based on the peak area at a specific wavelength absorbance, so the CA02 chicoric acid titer was calculated to be 12.5 mg / L based on the standard curve of chicoric acid.

Claims

1. A method for synthesizing chicoric acid metabolically engineered from glucose and tartaric acid in a yeast strain of Saccharomyces cerevisiae, comprising the following steps: 1) Selection of strains and plasmids: Escherichia coli DH5α was used for all plasmid construction and propagation; Saccharomyces cerevisiae CEN.PK2-1C was used as the starting strain; 2) DNA manipulation: 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. EpHTT and EpCAS from Echinacea were amplified using Echinacea cDNA as templates. The codon-optimized gene AtHCT was from Arabidopsis thaliana. The expression cassette promoters all used the strong promoters TPI1p, TEF1p, and PGKp to drive these genes, respectively. These candidate genes, promoters, or terminators were then cloned into helper plasmids using restriction ligation or Gibson assembly to obtain gene expression cassette plasmids. 3) Strain construction: 3.1) Using the CRISPR / Cas9 system, gene deletion and site-specific integration of DNA fragments were performed in Saccharomyces cerevisiae 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 of Saccharomyces cerevisiae CEN.PK2-1C. The resulting strain YT00:CEN.PK2-1C, IX1::TEFp-SpCas9-ADH2t was used as a host for DNA integration and biosynthetic pathway engineering. Equimolar amounts of the purified linearized fragments were co-transformed with the corresponding gRNA plasmids into the Saccharomyces cerevisiae strain 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 GreenTaqMix. Subsequently, correct clones were inoculated into YPD liquid medium, cultured overnight, serially passaged three times, and then plated on antibiotic-free plates to remove the gRNA vector. 3.2) Construction of a de novo p-coumaric acid synthesis pathway from glucose using the phenylalanine pathway: A pathway from phenylalanine to coumaric acid (CIA) was constructed in Saccharomyces cerevisiae by introducing the phenylalanine lyase AtPAL2 and the cinnamate hydroxylase AtC4H into the YT00 strain, resulting in strain YT01. Subsequently, the P450 reductase AtATR2 was introduced, and the native yeast cytochrome CYB5 was overexpressed to construct the biosynthetic pathway from CIA to p-coumaric acid (p-HCA), resulting in strain YT02. 3.3) Construction of a de novo chlorogenic acid pathway from glucose: Strain YC01 was generated by integrating quinate dehydrogenase EcYdiB, hydroxycinnamoyl-CoA quinoyltransferase CsHQT2, p-coumarate 3'-hydroxylase AtC3'H, cytochrome P450 reductase AtATR2, and 4-coumarate-CoA ligase At4CL1 into strain YT02. 3.4) Increasing chlorogenic acid production by releasing carbon flux in the shikimate pathway: Overexpression of ARO4 in the YC01 strain K229L mutants, which maximize carbon flux into the SA pathway and increase CGA production; Obtain strains YC02, YC01, △trp1::TEF1p-ARO4 K229L -CYC1t; 3.5) Optimizing L-phenylalanine branching and balancing the flux of p-HCA and QA to improve CGA yield: overexpressing Aro7 in the YC02 strain G141S , construct strain YC05, namely YC02, △ho-1::(HXT7t-ARO7 G141S -PGK1p)-(TPI1p-ARO3 K222L -TPI1t); Then, the single PHA2 overexpressing strain YC0701 was constructed, namely YC05,III1::GPM1p-PHA2-ADH1t; 3.6) Optimizing the supply of shikimate pathway precursors to increase CGA production: Based on the YC0701 strain, CRISPR-guided PYK1 D147N In vivo directed mutagenesis resulted in strain YC0702, namely YC0701, PYK11::PYK1 D147N ; After further overexpression of TKL1, strain YC0703, namely YC0702, XII5::PGK1p-TKL1-HXT7t; 3.7) Optimizing CGA biosynthesis by regenerating NADPH and adjusting the copy number of CGA biosynthesis genes: POS5 was overexpressed in strain YC0703 to promote intracellular NADPH production, thereby optimizing CGA biosynthesis. This resulted in strain YC0704 (YC0703, Δho-2::GPDp-POS5). Furthermore, CGA production was increased by integrating the CGA biosynthesis pathway genes AtC3H and CsHQT2, resulting in strain YC0707 (YC0704, X2::ENO2p). -HQT2-TPI1p-C3H; based on YC0707, the endogenous strong promoter pPGK1 was inserted as a means of overexpressing the gene ZWF1 to construct the overexpression strain YS04, namely YC0707, ZWF1p::PGK1p; the GAL1p-RgTAL-CYCt expression cassette was integrated into the GAL80 locus of YS04 to obtain YS08, namely YS04, ΔGAL80::GAL1p-RgTAL-CYC1t. YS08 is the starting strain for constructing a chicoric acid-producing strain; 3.8) Synthesizing chicoric acid by introducing a chicoric acid synthesis module; Using the pRS425-pTPI1-AtHCT plasmid as a template, the TPI1p-AtHCT-TPI1t-TEF1p expression cassette was amplified by PCR. The plasmid pRS425-TEF1p-AtF6'H1 was cut with BamHⅠ and XholⅠ restriction enzymes to obtain the linearized pRS425 plasmid. The homologous sequences at both ends of the linearized vector were introduced into the 5' end of the forward and reverse amplification primers of the inserted fragment EpHTT, so that the 5' and 3' ends of the amplified inserted fragment had and linearized sequences, respectively. The two ends of the cloning vector correspond to the same homologous sequence, and then the fragment after enzyme digestion is homologously recombined with the tartrate hydroxycinnamoyltransferase EpHTT with the corresponding homologous arm. The constructed plasmid is transformed into Escherichia coli DH5α to clone the plasmid to obtain the plasmid pRS425-pTEF1-EpHTT. Using the pRS425-pTEF1-EpHTT plasmid as a template, the TEF1p-EpHTT-TEF1t expression cassette is amplified by PCR. Similarly, the plasmid pRS425-P GK1p-At4CL1 was used to obtain the pRS425 linearized plasmid after enzyme digestion. The enzyme-digested fragment was homologously recombined with Echinacea purpurea chicoric acid synthase EpCAS with the corresponding homology arms. The constructed plasmid was transformed into Escherichia coli DH5α to clone the plasmid and obtain the plasmid pRS425-pPGK1-EpCAS. The TEF1t-PGK1p-EpCAS-HXT7t expression cassette was amplified by PCR using the pRS425-pPGK1-EpCAS plasmid as a template. Since the three expression cassettes each contained 500 bp complementary DNA sequences, the TEF1t-PGK1p-EpCAS-HXT7t expression cassette was amplified by PCR. Sequence, equal amounts of linearized fragments and gRNA were co-transformed into Saccharomyces cerevisiae cells by LiAc / ssDNA / PEG transformation method, and the AtHCT gene from Arabidopsis thaliana and the EpHTT and EpCAS genes from Echinacea purpurea were introduced into the X4 site of YS08 strain using CRISPR / Cas9 technology to obtain strain CA01, namely YS08,X4::(TPI1p-AtHCT-TPI1t)-(TEF1p-EpHTT-TEF1t)-(PGK1p-EpCAS-HXT7t); 3.9) Increasing chicoric acid production by increasing the copy number of the chicoric acid module: The copy number of each of the three genes (hydroxycinnamoyltransferase HCT, Echinacea hydroxycinnamoyl-CoA:tartrate hydroxycinnamoyltransferase EpHTT, and Echinacea chicoric acid synthase EpCAS) in the CA01 strain was increased to two to construct CA02, namely CA01, XVI::(TPI1p-AtHCT-TPI1t)-(TEF1p-EpHTT-TEF1t)-(PGK1p-EpCAS-HXT7t).

2. An application of the engineered strain CA02 constructed by the method according to claim 1 in the fermentation production of chicoric acid, characterized in that: The steps include: The engineered strain CA02 was inoculated into YPD medium and cultured at 30°C and 220 rpm for 24 hours to obtain seed liquid; the initial bacterial concentration OD660 was inoculated into a triangular flask containing seed medium at 0.05, and 0.5 mL of 100 g / L tartaric acid solution was added to the triangular flask. The fermentation was carried out at 30°C and 220 rpm to obtain a fermentation liquid of chicoric acid; the YPD medium composition: 20 g / L glucose, 10 g / L yeast powder, and 20 g / L peptone.

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