Engineered saccharomyces cerevisiae strain for producing bakuchiol and construction method and application thereof

Abstract

The application discloses a brewing yeast engineering strain for producing bakuchiol as well as a construction method and application thereof. The engineering strain overexpresses a gene in a geranyl pyrophosphate synthesis pathway, a gene in a p-coumaric acid synthesis pathway and a mutant isopentenyl transferase PcPT07 coding gene; the engineering strain has the ability to synthesize bakuchiol from glucose as a single carbon source; the classification and naming of the engineering strain is Saccharomyces cerevisiae BAK07, and the preservation number is CGMCC NO.31081. The application realizes the de novo synthesis of bakuchiol in the brewing yeast through overexpression of the gene in the geranyl pyrophosphate synthesis pathway, the gene in the p-coumaric acid synthesis pathway and the mutant isopentenyl transferase PcPT07 and combining metabolic engineering and other strategies, and lays a foundation for producing bakuchiol through microbial fermentation.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and synthetic biology, specifically relating to a brewer's yeast that produces psoralen, its construction method, and its applications. Background Technology

[0002] Bakuchiol is a terpene phenolic compound extracted and isolated from the seeds of the plant Psoralea corylifolia L. Medik. It possesses a wide range of biological activities, including anticancer, antioxidant, antibacterial, and immunosuppressive effects. Bakuchiol can be used as a skin protectant, skin feel modifier, moisturizer, and antioxidant. Due to its retinol-like effects, milder action, and photostable properties, it has received increasing attention in the cosmetics industry.

[0003] Like most natural products, the production of psoralen primarily relies on extraction from plants. However, psoralen coexists with other coumarin compounds that can promote skin cancer, such as psoralen and isopsoralen, placing higher demands on downstream purification steps. Furthermore, factors such as the long plant growth cycle, environmental and climatic requirements, and unpredictable natural factors pose challenges to the supply of psoralen. From an organic synthesis perspective, although the total synthesis of psoralen has been reported, the stringent reaction conditions and the use of toxic chemical reagents limit its industrial production. Therefore, preparing high-purity and high-content psoralen remains a significant challenge.

[0004] The heterologous and efficient synthesis of psoralen using microorganisms is a sustainable, environmentally friendly, and highly attractive approach with considerable economic and social benefits. However, the biosynthetic pathway of psoralen remains incompletely elucidated, with key enzymes still unclear. Therefore, there is an urgent need for a psoralen-producing microbial cell and its construction method that can address these issues. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a *Saccharomyces cerevisiae* engineered strain that produces psoralen, its construction method, and its applications. The *Saccharomyces cerevisiae* engineered strain of this invention can generate psoralen using p-coumaric acid (3-(4-hydroxyphenyl)-2-acrylic acid) as a substrate through catalytic trans-geranylation and coupled decarboxylation. Furthermore, the *Saccharomyces cerevisiae* engineered strain of this invention can also synthesize psoralen de novo using glucose as a single carbon source.

[0006] The technical solution adopted in this invention is as follows:

[0007] (a) An engineered strain of *Saccharomyces cerevisiae* that produces bakuchiol

[0008] The engineered strain of *Saccharomyces cerevisiae* overexpresses the gene encoding the geranyl pyrophosphate synthesis pathway, the gene encoding the coumaric acid synthesis pathway, and the gene encoding the mutant isopentenyltransferase PcPT07.

[0009] The genes governing the gerany-based pyrophosphate synthesis pathway include the gene encoding acetyl-CoA thiolase / HMG-CoA reductase EfMvaE from Enterococcus faecalis and the mutant mevalonate synthase EfMvaS. A110G The gene encoding the yeast isopentenyl diphosphate isomerase IDI1, mevalonate phosphate kinase ERG8, mevalonate diphosphate decarboxylase ERG19, mevalonate kinase ERG12, and geranyl pyrophosphate synthase mutant ERG20. F96W / N127W Encoding genes.

[0010] The genes governing the p-coumaric acid synthesis pathway include the AtPAL2 gene encoding phenylalanine ammonia-lyase, the AtC4H gene encoding cinnamic acid-4-hydroxylase, and the AtATR2 gene encoding P450 reductase from Arabidopsis thaliana; the FjTAL gene encoding tyrosine ammonia-lyase from Flavobacterium johnsonii; and the mutant 3-deoxy-D-arabinohepenolate-7-phosphate synthase ARO4 from Saccharomyces cerevisiae. K229L Encoding gene and cladistic acid mutase ARO7 G141S Encoding genes.

[0011] The engineered strain of *Saccharomyces cerevisiae* has the ability to synthesize psoralen de novo using glucose as a single carbon source, that is, the ability to generate psoralen de novo using glucose as a substrate.

[0012] Specifically, the engineered strain of Saccharomyces cerevisiae generates psoralen through trans-geranylation of coumaric acid via an intermediate product followed by a coupling decarboxylation reaction.

[0013] The mutant isopentenyltransferase PcPT07 is used to catalyze the trans-geranylation and coupled decarboxylation of p-coumaric acid. The mutant isopentenyltransferase PcPT07 is a truncated isopentenyltransferase PcPT07-FL, and the truncation range is the first 84 amino acid sequences of the N-terminus of the isopentenyltransferase PcPT07-FL. The isopentenyltransferase PcPT07-FL is derived from psoralea corylifolia.

[0014] The engineered Saccharomyces cerevisiae strain was introduced into the mutant isopentenyltransferase PcPT07 encoding gene via at least one of the following integration methods:

[0015] a: An expression cassette containing the gene encoding the mutant isopentenyltransferase PcPT07 is integrated into the genome of the engineered strain of *Saccharomyces cerevisiae*.

[0016] b: The genome of the engineered Saccharomyces cerevisiae strain integrates an expression cassette containing 1 to 3 fusion gene fragments, wherein the fusion gene fragments are mainly encoded by the mutant isopentenyltransferase PcPT07 gene via a linker peptide gene and the geranyl pyrophosphate synthase mutant ERG20. F96W / N127W It is obtained by linking coding genes.

[0017] The amino acid sequence of the mutant isopentenyltransferase PcPT07 is shown in SEQ ID NO.1.

[0018] The nucleotide sequence of the gene encoding the mutant isopentenyltransferase PcPT07 is shown in SEQ ID NO.2; the amino acid sequence of the isopentenyltransferase PcPT07-FL is shown in SEQ ID NO.15.

[0019] The nucleotide sequence of the fusion gene fragment is shown in SEQ ID NO.16; the nucleotide sequence of the gene encoding the linker peptide is shown in SEQ ID NO.17; and the amino acid sequence of the linker peptide is shown in SEQ ID NO.18.

[0020] The engineered strain of brewer's yeast has also been modified in at least one of the following ways:

[0021] a) In the genome of the engineered Saccharomyces cerevisiae strain, the endogenous pyruvate decarboxylase PDC5 encoding gene and the phenylpyruvate decarboxylase ARO10 encoding gene were knocked out.

[0022] b) In the genome of the engineered Saccharomyces cerevisiae strain, the promoter of the farnesyl pyrophosphate synthase ERG20 gene was replaced in situ with the promoter of the squalene epoxidase ERG1 gene.

[0023] The engineered strain of *Saccharomyces cerevisiae* is classified as *Saccharomyces cerevisiae* BAK07, with accession number CGMCC NO.31081. The depository is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is June 25, 2024. The *Saccharomyces cerevisiae* BAK07 strain originated from *Saccharomyces cerevisiae* BY4742. The genome of *Saccharomyces cerevisiae* BAK07 integrates the geranyl pyrophosphate synthesis pathway gene, the coumaric acid synthesis pathway gene, the mutant isopentenyltransferase PcPT07 encoding gene, and the fusion gene fragment. Furthermore, the genome of *Saccharomyces cerevisiae* BAK07 also has the pyruvate decarboxylase PDC5 encoding gene and the phenylpyruvate decarboxylase ARO10 encoding gene knocked out. The promoter of the farnesyl pyrophosphate synthase ERG20 encoding gene in the *Saccharomyces cerevisiae* BAK07 genome has been replaced in situ with the promoter of the squalene epoxidase ERG1 encoding gene.

[0024] (II) A method for constructing an engineered strain of *Saccharomyces cerevisiae* that produces bakuchiol.

[0025] The construction method specifically involves: constructing multiple expression cassettes, integrating each expression cassette into different sites in the genome of Saccharomyces cerevisiae using homologous recombination technology to obtain a first strain, and using the first strain as the engineered strain;

[0026] The expression cassette includes a first expression cassette, a second expression cassette, a third expression cassette, a fourth expression cassette, and a fifth expression cassette; the first expression cassette includes the gene encoding phenylalanine ammonia-lyase AtPAL2, the gene encoding cinnamic acid-4-hydroxylase AtC4H, and the gene encoding P450 reductase AtATR2; the second expression cassette includes the gene encoding tyrosine ammonia-lyase FjTAL and the mutant 3-deoxy-D-arabinohepenolate-7-phosphate synthase ARO4. K229L Encoding gene and cladistic acid mutase ARO7 G141S The third expression cassette contains the gene encoding acetyl-CoA thiolase / HMG-CoA reductase EfMvaE and the mutant mevalonate synthase EfMvaS. A110G The fourth expression cassette contains the encoding genes for mevalonate kinase ERG12, mevalonate phosphate kinase ERG8, and mevalonate diphosphate decarboxylase ERG19; the fifth expression cassette contains the geranyl pyrophosphate synthase mutant ERG20. F96W / N127W The encoding gene and the mutant isopentenyltransferase PcPT07 encoding gene.

[0027] The construction method further includes the following steps:

[0028] The second strain was obtained by knocking out the PDC5 gene encoding endogenous pyruvate decarboxylase and the ARO10 gene encoding phenylpyruvate decarboxylase in the first strain using homologous recombination technology.

[0029] By using homologous recombination technology, the promoter of the farnesyl pyrophosphate synthase ERG20 gene in the genome of the second strain was replaced in situ with the promoter of the squalene epoxidase ERG1 gene to obtain the third strain.

[0030] The mutant isopentenyltransferase PcPT07 encoding gene was linked to the geranyl pyrophosphate synthase mutant ERG20 gene via a linker peptide. F96W / N127W The coding genes are linked to obtain a fusion gene fragment; an expression cassette containing a copy of the fusion gene fragment is constructed based on the fusion gene fragment; the expression cassette containing 1 to 3 copies of the fusion gene fragment is integrated into the genome of a third strain using homologous recombination technology to obtain a fourth strain, which is then used as the engineered strain of *Saccharomyces cerevisiae*.

[0031] (III) Application of an engineered strain of *Saccharomyces cerevisiae* that produces bakuchiol

[0032] This invention is used to produce psoralen by catalytic trans-geranylation of p-coumaric acid and coupling decarboxylation, using glucose as a substrate and p-coumaric acid, a product of glucose, as an intermediate.

[0033] (iv) A method for producing bakuchiol using engineered strains of Saccharomyces cerevisiae that produce bakuchiol.

[0034] The engineered strain of *Saccharomyces cerevisiae* was cultured in a fermentation medium to obtain a fermentation culture. The fermentation culture was then extracted and separated to obtain psoralen.

[0035] The beneficial effects of this invention are as follows:

[0036] 1. This invention solves the key enzyme required for the synthesis of psoralen by strengthening the mevalonic acid pathway in Saccharomyces cerevisiae and integrating the gene encoding the mutant isopentenyltransferase PcPT07, thus achieving the effect of synthesizing psoralen through microbial use of exogenous coumaric acid.

[0037] 2. This invention achieves the effect of de novo synthesis of bakuchiol by microorganisms using glucose as a single carbon source by further integrating endogenous and exogenous genes into the genome of Saccharomyces cerevisiae.

[0038] 3. This invention enhances the relevant reaction pathways by knocking out endogenous genes, replacing endogenous promoters in situ, and integrating fusion proteins, thereby improving the efficiency and yield of de novo synthesis of bakuchiol by microorganisms. The shake-flask fermentation yield of the engineered strain Saccharomyces cerevisiae BAK07 obtained by the above modification methods can reach 9.27 mg / L. Attached Figure Description

[0039] Figure 1 A schematic diagram of the biosynthetic pathway of psoralen;

[0040] Figure 2 This is a liquid phase diagram of the in vivo catalytic synthesis of psoralen;

[0041] Figure 3 This is a liquid phase diagram of the in vitro catalytic synthesis of psoralen;

[0042] Figure 4 This is the LC-MS analysis chromatogram of psoralen in anion mode. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] Unless otherwise specified, in this invention, except for the mutant isopentenyltransferase PcPT07 (obtained by truncating the original isopentenyltransferase PcPT07-FL to its N-terminus), other mutant proteins are named in the format of "protein name first mutation site / second mutation site / ... / Nth mutation site". The symbol " / " between two mutation sites represents "and". For example, the geranyl pyrophosphate synthase mutant ERG20. F96W / N127W This indicates that it is derived from gerany pyrophosphate synthase ERG20 via the F96W and N127W mutations. Furthermore, protein mutation types are named according to the format "original amino acid - site number - mutated amino acid". For example, the F96W mutation indicates that at the 96th amino acid position of the protein, the original phenylalanine (F) is replaced by tryptophan (W).

[0045] This invention provides a *Saccharomyces cerevisiae* engineered strain that produces psoralen. This strain has the ability to synthesize the intermediate p-coumaric acid using glucose as a substrate, and to generate psoralen through trans-geranylation and coupling decarboxylation of p-coumaric acid. Figure 1 ).

[0046] This engineered strain of *Saccharomyces cerevisiae* overexpresses genes encoding the gerany pyrophosphate synthesis pathway, the coumaric acid synthesis pathway, and the mutant isopentenyltransferase PcPT07.

[0047] The mutant isopentenyltransferase PcPT07 encoding gene expresses the mutant isopentenyltransferase PcPT07, which catalyzes the trans-geranylation of p-coumaric acid followed by decarboxylation to generate psoralen. The amino acid sequence of the mutant isopentenyltransferase PcPT07 is shown in SEQ ID NO.1. The mutant isopentenyltransferase PcPT07 is a truncated version of isopentenyltransferase PcPT07, with the truncated portion being the first 84 amino acids from the N-terminus of the protein. The amino acid sequence of isopentenyltransferase PcPT07-FL is shown in SEQ ID NO.15.

[0048] Among them, the genes involved in the p-coumaric acid synthesis pathway include:

[0049] 1) Expresses FjTAL, a tyrosine ammonia-lyase derived from Flavobacterium johnsonii. The GenBanK number for the FjTAL encoding gene is WP_012023194.1; it is used to catalyze ① the process of converting tyrosine to p-coumaric acid. Figure 1 );

[0050] 2) It expresses the Arabidopsis-derived phenylalanine ammonia-lyase AtPAL2, whose GenBanK number is NM_115186.4; it is involved in catalyzing the process ② from phenylalanine to p-coumaric acid. Figure 1 );

[0051] 3) It expresses cinnamic acid-4-hydroxylase AtC4H derived from Arabidopsis thaliana. The GenBanK number of the AtC4H encoding gene is NM_128601.3; it is used to catalyze the process ② from phenylalanine to p-coumaric acid. Figure 1 );

[0052] 4) Expresses Arabidopsis-derived P450 reductase AtATR2, the GenBanK number of the AtATR2 encoding gene is NM_119167.4; it is involved in catalyzing the process ② from phenylalanine to p-coumaric acid. Figure 1 );

[0053] 5) Expressing the mutant 3-deoxy-D-arabinohepetulose-7-phosphate synthase ARO4 derived from Saccharomyces cerevisiae. K229L Encoding gene and cladistic acid mutase ARO7 G141S Encoding gene, ARO4 K229L The nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; ARO7 G141S The nucleotide sequence of the encoding gene is shown in SEQ ID NO.4, and it is used to improve the efficiency of glucose conversion to tyrosine and glucose conversion to phenylalanine.

[0054] Among them, the genes involved in the gerany pyrophosphate synthesis pathway include:

[0055] 1) Expression of EfMvaE, an acetyl-CoA thiodiolase / HMG-CoA reductase derived from Enterococcus faecalis, with the GenBanK number KX064239; used to increase the content of geranyl pyrophosphate (GPP). Figure 1 );

[0056] 2) Expressing a mutant mevalonate synthase EfMvaS derived from Enterococcus faecalis. A110G EfMvaS A110G The nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; used to increase the content of geraniol pyrophosphate ( Figure 1 );

[0057] 3) Expression of the following mutants from *Saccharomyces cerevisiae*: mevalonate phosphate kinase ERG8, mevalonate diphosphate decarboxylase ERG19, mevalonate kinase ERG12, isopentenyl diphosphate isomerase IDI1, and geranyyl pyrophosphate synthase ERG20. F96W / N127W Since yeast has a low endogenous geranyl pyrophosphate content, the present invention introduces the above-mentioned enzyme to increase the geranyl pyrophosphate content.

[0058] By integrating the above genes into the starting strain of Saccharomyces cerevisiae, an engineered strain of Saccharomyces cerevisiae was constructed. This engineered strain of Saccharomyces cerevisiae has the ability to synthesize bakuchiol de novo using glucose as a substrate.

[0059] Furthermore, based on the above-mentioned engineered strains, knocking out the PDC5 gene encoding endogenous pyruvate decarboxylase and the ARO10 gene encoding phenylpyruvate decarboxylase in Saccharomyces cerevisiae can enhance the fermentation production of bakuchiol by increasing the efficiency of glucose conversion to tyrosine and glucose conversion to phenylalanine.

[0060] Furthermore, since the endogenous farnesyl diphosphate synthase ERG20 in Saccharomyces cerevisiae consumes gerany pyrophosphate, and knocking out ERG20 is lethal, the ability of the engineered strain to produce bakuchiol can be improved by replacing the ERG20 promoter with the ERG1 promoter through in-situ substitution, based on the aforementioned engineered strain.

[0061] Furthermore, to enhance the catalytic activity of the mutant isopentenyltransferase PcPT07, the geranyyl pyrophosphate synthase mutant ERG20 was linked via a linker peptide (GGGGS). F96W / N127W It was fused to the N-terminus of the mutant isopentenyltransferase PcPT07 to generate the fusion protein ERG20. F96W / N127W -GGGGS-PcPT07. The fusion protein ERG20 is integrated via a multi-copy integration method.F96W / N127W The integration of the -GGGGS-PcPT07 encoding gene into the genome of the above-mentioned engineered strain can enhance the ability of the engineered strain to produce psoralen through fermentation.

[0062] This invention also provides an engineered strain of *Saccharomyces cerevisiae*: *Saccharomyces cerevisiae* BAK07. *Saccharomyces cerevisiae* BAK07 is derived from *Saccharomyces cerevisiae* BY4742 and integrates genes encoding the geranyl pyrophosphate synthesis pathway, the coumaric acid synthesis pathway, the mutant isopentenyltransferase PcPT07 gene, and three fusion gene fragments. The genome of *Saccharomyces cerevisiae* BAK07 also has the genes encoding pyruvate decarboxylase PDC5 and phenylpyruvate decarboxylase ARO10 knocked out. Furthermore, the promoter of the farnesyl pyrophosphate synthase ERG20 gene in the *Saccharomyces cerevisiae* BAK07 genome has been replaced in situ with the promoter of the squalene epoxidase ERG1 gene. This strain can efficiently synthesize bakuchiol, achieving a bakuchiol yield of 9.28 mg / L during shake-flask fermentation.

[0063] In this invention, the gene encoding the geranyl pyrophosphate synthesis pathway, the gene encoding the coumaric acid synthesis pathway, and the gene encoding the mutant isopentenyltransferase PcPT07 were integrated into different sites on the genome of Saccharomyces cerevisiae.

[0064] In this invention, the method for constructing the expression box and the method for integration and recombination can both adopt conventional methods well known to those skilled in the art.

[0065] This invention provides a method for constructing an engineered strain capable of producing psoralen using exogenous p-coumaric acid, specifically as follows:

[0066] Multiple expression cassettes were constructed using conventional methods well-known to those skilled in the art. These cassettes were then integrated into different sites in the genome of *Saccharomyces cerevisiae* using homologous recombination technology, resulting in an engineered strain capable of producing psoralen from exogenous p-coumaric acid. The expression cassettes included:

[0067] The third expression cassette includes constitutive promoters FBA1p, PGK1p, and TEF1p; terminators VPS13t, TEF2t, and CYC1t; and mevalonate pathway enzymes EfMvaE and EfMvaS. A110G IDI1 and the upstream and downstream homologous arms US_911b and DS_911b of the insertion site are used to enhance the mevalonate pathway, thereby increasing GPP content, specifically:

[0068] US_911b / FBA1p-EfMvaE-VPS13t / PGK1p-EfMvaS A110G -TEF2t / TEF1p-IDI1-CYC1t / DS_911b;

[0069] The fourth expression cassette includes constitutive promoters TPI1p, TEF2p, and TEF1p; terminators GND2t, ENO2t, and CYC1t; enzymes of the mevalonate pathway ERG12, ERG8, and ERG19; and upstream and downstream homologous arms US_308a and DS_308a at the insertion site, specifically:

[0070] US_308a / TPI1p-ERG12-GND2t / TEF2p-ERG8-ENO2t / TEF1p-ERG19-CYC1t / DS_308a;

[0071] The fifth expression cassette includes constitutive promoters TDH3p and TEF2p; terminators TEF1t and ENO2t; mutant isopentenyltransferase PcPT07; and geranyl pyrophosphate synthase mutant ERG20. F96W / N127W The upstream and downstream homologous arms US_YPRCδ15 and DS_YPRCδ15, along with the insertion site, are used to catalyze the synthesis of psoralen from p-coumaric acid and the GPP precursor. Specifically:

[0072] US_YPRCδ15 / TDH3p-PcPT07-TEF1t / TEF2p-ERG20 F96W / N127W -ENO2t / DS_YP RCδ15.

[0073] This invention also provides a method for constructing an engineered strain capable of de novo synthesis of psoralen using glucose as a single carbon source, specifically as follows:

[0074] Multiple expression cassettes were constructed using conventional methods well-known to those skilled in the art. These cassettes were then integrated into different sites in the genome of *Saccharomyces cerevisiae* using homologous recombination technology, resulting in the first strain capable of de novo synthesis of psoralen using glucose as a single carbon source. The expression cassettes included:

[0075] The first expression cassette includes constitutive promoters ENO2p, TDH3p, and PGK1p; terminators CYC1t, TEF1t, and TEF2t; enzymes AtPAL2, AtC4H, and AtATR2 for the coumaric acid synthesis pathway; and upstream and downstream homologous arms US_911b and DS_911b at the insertion site, used to achieve de novo synthesis of the key precursor coumaric acid in Saccharomyces cerevisiae. Specifically:

[0076] US_911b / ENO2p-AtPAL2-CYC1t / TDH3p-AtC4H-TEF1t / PGK1p-AtATR2-TE F2t / DS_911b;

[0077] The second expression cassette includes constitutive promoters TDH3p, TEF2p, and TPI1p; terminators TEF1t, ENO2t, and GND2t; and coumarate synthesis pathway enzymes FjTAL and ARO4. K229L ARO7 G141S The upstream and downstream homologous arms US_1014a and DS_1014a of the insertion site are used to relieve the negative feedback inhibition in yeast synthesis of the amino acids phenylalanine and tyrosine, and to increase the yield of the key precursor coumaric acid. Specifically:

[0078] US_1014a / TDH3p-FjTAL-TEF1t / TEF2p-ARO4 K229L -ENO2t / TPI1p-ARO7 G141S -GND2t / DS_1014a;

[0079] The third expression cassette includes constitutive promoters FBA1p, PGK1p, and TEF1p; terminators VPS13t, TEF2t, and CYC1t; and mevalonate pathway enzymes EfMvaE and EfMvaS. A110G The IDI1 and upstream and downstream homologous arms US_416d and DS_416d of the insertion site (the integration site of this expression cassette is different from the integration site of the third expression cassette mentioned above), specifically:

[0080] US_416d / FBA1p-EfMvaE-VPS13t / PGK1p-EfMvaS A110G -TEF2t / TEF1p-IDI1-CYC1t / DS_416d;

[0081] The fourth expression cassette includes constitutive promoters TPI1p, TEF2p, and TEF1p; terminators GND2t, ENO2t, and CYC1t; enzymes of the mevalonate pathway ERG12, ERG8, and ERG19; and upstream and downstream homologous arms US_308a and DS_308a at the insertion site, specifically:

[0082] US_308a / TPI1p-ERG12-GND2t / TEF2p-ERG8-ENO2t / TEF1p-ERG19-CYC1t / DS_308a;

[0083] The fifth expression cassette includes constitutive promoters TDH3p and TEF2p; terminators TEF1t and ENO2t; mutant isopentenyltransferase PcPT07; and geranyl pyrophosphate synthase mutant ERG20. F96W / N127W The upstream and downstream homologous arms of the insertion site, US_YPRCδ15 and DS_YPRCδ15, are specifically as follows:

[0084] US_YPRCδ15 / TDH3p-PcPT07-TEF1t / TEF2p-ERG20 F96W / N127W -ENO2t / DS_YP RCδ15.

[0085] This invention also provides a method for modifying an engineered strain capable of de novo synthesis of psoralen using glucose as a single carbon source to obtain a high-yield psoralen-producing strain. This modification method comprises three steps, each independently effective, and any step can be selected for execution based on the experimental objective. Furthermore, to achieve more efficient experimental results, these steps can be performed simultaneously or sequentially according to the specific requirements of the experimental design.

[0086] Using the engineered strain that can utilize glucose as a single carbon source to synthesize psoralen de novo as the first strain, the modification method includes the following three steps:

[0087] Step 1: Knock out the PDC5 gene encoding endogenous pyruvate decarboxylase and the ARO10 gene encoding phenylpyruvate decarboxylase from the genome of the target strain using homologous recombination technology;

[0088] Step 2: Replace the promoter of the farnesyl pyrophosphate synthase ERG20 gene in the genome of the target strain in situ with the promoter of the squalene epoxidase ERG1 gene using homologous recombination technology; this step can be achieved based on the following expression cassette: US_ERG20-ERG1p-DS_ERG20;

[0089] Step 3: The gene encoding the mutant isopentenyltransferase PcPT07 is ligated to the gene encoding the geranyl pyrophosphate synthase mutant ERG20 via a linker. F96W / N127W The fusion gene fragment is obtained from the 5' end of the coding gene; an expression cassette containing the fusion gene fragment is constructed based on the fusion gene fragment, and the expression cassette containing 1 to 3 fusion gene fragments is integrated into the genome of the target strain using homologous recombination technology.

[0090] The nucleotide sequence of the gene encoding the linker peptide is GGTGGTGGCGGTTCC, as shown in SEQ ID NO.17. The amino acid sequence of the linker peptide is GGGGS, as shown in SEQ ID NO.18.

[0091] Step three can be implemented based on any of the following expression boxes:

[0092] Single-copy expression cassette, containing one fusion gene fragment:

[0093] US_XII-2 / PGK1p-ERG20F96W / N127W-GGGGS-PcPT07t-TEF2t / DS_XII-2;

[0094] The dual-copy expression cassette contains two fusion gene fragments:

[0095] US_XII-2 / PGK1p-ERG20F96W / N127W-GGGGS-PcPT07t-TEF2t / FBA1p-ER G20F96W / N127W-GGGGS-PcPT07t-VPS13 / DS_XII-2;

[0096] The three-copy expression cassette contains three fusion gene fragments:

[0097] US_XII-2 / PGK1p-ERG20F96W / N127W-GGGGS-PcPT07t-TEF2t / FBA1p-ER G20F96W / N127W-GGGGS-PcPT07t-VPS13 / ENO2p-ERG20F96W / N127W-GGGG S-PcPT07t-CYC1t / DS_XII-2.

[0098] To more clearly illustrate the objectives and technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without any inventive effort are within the scope of protection of this invention. In the embodiments of this invention, the starting strain *Saccharomyces cerevisiae* BY4742 was obtained through commercial purchase.

[0099] Example 1

[0100] This embodiment first uses *Saccharomyces cerevisiae* BY4742 (with a pre-integrated Cas9 gene expression cassette in its genome) as the starting strain. A *Saccharomyces cerevisiae* strain with enhanced mevalonic acid pathway was constructed to increase the supply of geranyl pyrophosphate (GPP), resulting in the high-yielding *Saccharomyces cerevisiae* BAK01. Next, a mutant isopentenyltransferase PcPT07 was introduced into the genome of *Saccharomyces cerevisiae* BAK01, resulting in *Saccharomyces cerevisiae* BAK02. This was used to verify the function of the mutant isopentenyltransferase PcPT07 in catalyzing the trans-geranylation of coumaric acid and its subsequent decarboxylation to generate psoralen.

[0101] Construction of a high-geraniol pyrophosphate-producing Saccharomyces cerevisiae BAK01

[0102] Gene fragments were amplified and integrated using PCR: Using the *Saccharomyces cerevisiae* BY4742 (GenBank: GCA_003086655.1) genome as a template, homologous arms upstream and downstream of the 911b and 308a integration sites (US_911b, DS_911b, US_308a, DS_308a, respectively) were amplified by PCR; using the *Saccharomyces cerevisiae* BY4742 genome as a template, fragments of the FBA1 promoter, VPS13 terminator, PGK1 promoter, TEF2 terminator, TEF1 promoter, CYC1 terminator, TPI1 promoter, GND2 terminator, TEF2 promoter, ENO2t terminator, TEF1 promoter, and CYC1 terminator were amplified by PCR; using the *Saccharomyces cerevisiae* BY4742 genome as a template, gene fragments of IDI1, ERG12, ERG8, and ERG19 were amplified by PCR. EfMvaE and EfMvaS were obtained through gene synthesis. A110G Gene fragments. The above fragments were assembled by fusion PCR and ligated into the pTA2 vector via TA cloning. After sequencing confirmed the integrated fragments were correct, the following two corresponding gene expression cassettes were obtained:

[0103] Third expression box:

[0104] US_911b / FBA1p-EfMvaE-VPS13t / PGK1p-EfMvaS A110G -TEF2t / TEF1p-IDI1-CYC1t / DS_911b;

[0105] Fourth expression box:

[0106] US_308a / TPI1p-ERG12-GND2t / TEF2p-ERG8-ENO2t / TEF1p-ERG19-CYC1t / US_308a.

[0107] The above expression cassettes were transformed into Saccharomyces cerevisiae BY4742 along with sgRNA plasmids (sgRNA-911b and sgRNA-308a), respectively (note that after integration into the third expression cassette, it was then integrated into the fourth expression cassette). After verification of correct integration by colony PCR, the enhanced mevalonic acid pathway engineered Saccharomyces cerevisiae strain BAK01 was obtained.

[0108] Validation of the mutant isopentenyltransferase PcPT07

[0109] In this embodiment, the conserved motifs of isopentenyltransferases, namely DxxxD, YxxxK, and DxxDxxGD, were first used to screen candidate genes from the transcriptome data of Psoralea corylifolia plants. After experimental verification, the isopentenyltransferase PcPT07-FL, which can successfully catalyze the transgermination of coumaric acid and the coupling decarboxylation to generate psoralen, was obtained. The original amino acid sequence of isopentenyltransferase PcPT07-FL is shown in SEQ ID NO.15.

[0110] Next, RNA was extracted from *Psoralea corylifolia* plants and reverse transcribed into cDNA. The mutant isopentenyltransferase PcPT07 gene fragment (sequence shown in SEQ ID NO.2), truncated 84 amino acids at the N-terminus, was amplified by PCR. Truncation of the N-terminal signal peptide significantly enhanced the catalytic activity of the isopentenyltransferase. The signal peptide of the isopentenyltransferase PcPT07-FL was predicted using the bioinformatics analysis website TargetP 2.0. Using the *Saccharomyces cerevisiae* BY4742 genome as a template, multiple primers were designed to clone and construct the farnesyl synthase mutant ERG20. F96W / N127W Gene fragments were amplified using the *Saccharomyces cerevisiae* BY4742 genome as a template. Homologous arms upstream and downstream of the YPRCδ15 integration site (US_YPRCδ15 and DS_YPRCδ15) were amplified by PCR. The TDH3 promoter and TEF1 terminator fragments were also amplified by PCR using the *Saccharomyces cerevisiae* BY4742 genome as a template. These fragments were assembled via fusion PCR and ligated into the pTA2 vector using TA cloning. After successful sequencing of the integrated fragments, the corresponding gene expression cassettes were obtained.

[0111] Fifth Expression Box:

[0112] US_YPRCδ15 / TDH3p-PcPT07-TEF1t / TEF2p-ERG20 F96W / N127W -ENO2t / DS_YP RCδ15.

[0113] The fifth expression cassette, along with the sgRNA plasmid (sgRNA-YPRCδ15), was transformed into *Saccharomyces cerevisiae* BAK01. After colony PCR verification confirmed correct integration, *Saccharomyces cerevisiae* BAK02 was obtained.

[0114] The function of PcPT07 was verified by culturing Saccharomyces cerevisiae BAK01 and BAK02 separately and performing HPLC analysis on the fermentation products.

[0115] Culture of the strains. BAK01 and BAK02 were inoculated into 5 mL of YPD culture medium and cultured overnight at 30°C and 220 rpm, followed by OD... 600=0.2% was inoculated into 10 mL of YPD medium (100 mL Erlenmeyer flask) and cultured at 30℃ and 220 rpm for 2 days. The cells were collected by centrifugation (500 g, 4 min), resuspended in 1 mL of YPD medium containing 1 mM coumaric acid, and then cultured under the same conditions for 1 day. After the culture was completed, an equal volume of methanol was added to the medium and vortexed to mix. The resulting sample was centrifuged at 14000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was used for subsequent liquid chromatography analysis.

[0116] The fermentation products were analyzed by HPLC. The analytical method was as follows: a reversed-phase C18 column (5 μm, 4.6 × 150 mm, Agilent). The mobile phase consisted of 1‰ formic acid aqueous solution (A) and acetonitrile (B). The gradient elution program was as follows: 5%-35% B, 7 min; 35%-95% B, 3 min; 95% B, 5 min. The program run time was 5 min. The flow rate was 1.0 mL / min. p-coumaric acid (p-CA) was detected at 308 nm, and bakuchiol (BAK) was detected at 262 nm. The HPLC conditions in the following examples were consistent and will not be repeated hereafter. The HPLC results are as follows. Figure 2 As shown. The results indicate that PcPT07 can convert p-coumaric acid to produce a new product peak, which has the same retention time as the psoralen standard (e.g., ...). Figure 2 As shown). Rapid liquid chromatography-triple quadrupole mass spectrometry analysis, in anion mode, revealed the target compound to have a molecular weight of 455, consistent with the psoralen standard (as shown). Figure 4 (As shown). The product was confirmed to be psoralen.

[0117] To further confirm the function of PcPT07, engineered strains BAK01 and BAK02, cultured overnight, were placed in 200 mL of YPD medium (500 mL Erlenmeyer flask) and cultured at 30℃ and 220 rpm for 3 days. The bacterial pellet was then centrifuged and microsomes were extracted. The extracted microsomes were characterized in vitro. The 100 μL reaction system was as follows: 100 mM Tris HCl (pH 7.0), 100 μM p-coumaric acid, 80 μg microsomes, 200 μM GPP, and 10 mM MgCl2. The in vitro reaction conditions were 30℃ for 1 h. After the reaction, an equal volume of methanol was added to stop the reaction, the mixture was vortexed, centrifuged at maximum speed for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane for liquid chromatography analysis. The results are as follows: Figure 3 As shown in the figure. The results indicate that, similar to the in vivo analysis, PcPT07 can convert p-coumaric acid to produce a new product peak, which has the same retention time and UV absorption as the psoralen standard.

[0118] In summary, by introducing the mutant isopentenyltransferase PcPT07 derived from psoralen, the biosynthetic pathway of psoralen was improved, and the mutant isopentenyltransferase PcPT07, capable of catalyzing the trans-geranylation of coumaric acid and subsequent decarboxylation to generate psoralen, was identified. Thus, the biosynthetic pathway of psoralen has been fully elucidated (e.g., Figure 1 (As shown).

[0119] Example 2

[0120] To achieve de novo synthesis of bakuchiol, this embodiment uses Saccharomyces cerevisiae BY4742 (with the Cas9 gene expression cassette pre-integrated in its genome) as the starting strain to construct Saccharomyces cerevisiae BAK03, which can stably produce bakuchiol.

[0121] AtPAL2, AtC4H, AtATR2, and FjTAL gene fragments were obtained through gene synthesis. Using the *Saccharomyces cerevisiae* BY4742 genome as a template, multiple primers were designed for cloning, and the mutant ARO4 was constructed. K229L and ARO7 G141S Gene fragments: Using the *Saccharomyces cerevisiae* BY4742 genome as a template, PCR amplification was performed on the upstream and downstream homologous arms (US_1014a, DS_1014a, US_416d, DS_416d) of the integration sites at 1014a and 416d, each approximately 1 kbp in length. Using the *Saccharomyces cerevisiae* BY4742 genome as a template, PCR amplification was performed on the 853 bp fragment before the start codon of the PDC5 gene (US_PDC5) and the 617 bp fragment after the stop codon (DS_PDC5). The ARO10 gene fragment was amplified on the 451 bp fragment before the start codon (US_ARO10) and the 481 bp fragment after the stop codon (DS_ARO10). Using the *Saccharomyces cerevisiae* BY4742 genome as a template, PCR amplification was performed on the ENO2 promoter and TEF2 terminator fragments. These fragments were then assembled with the fragments mentioned above via fusion PCR and ligated into the pTA2 vector using TA cloning. After the integrated fragment was sequenced without errors, the corresponding gene expression cassettes and repair templates for PDC5 and ARO10 were obtained:

[0122] First Expression Box:

[0123] US_911b / ENO2p-AtPAL2-CYC1t / TDH3p-AtC4H-TEF1t / PGK1p-AtATR2-TE F2t / DS_911b;

[0124] Second expression box:

[0125] US_1014a / TDH3p-FjTAL-TEF1t / ENO2t-ARO4 K229L -TEF2p / TPI1p-ARO7G141S -GND2t / DS_1014a;

[0126] Third expression box:

[0127] US_416d / FBA1p-EfMvaE-VPS13t / PGK1p-EfMvaS A110G -TEF2t / TEF1p-IDI1-CYC1t / DS_416d;

[0128] PDC5 repair template:

[0129] US_PDC5 / DS_PDC5;

[0130] ARO10 Repair Template:

[0131] US_ARO10 / DS_ARO10;

[0132] Furthermore, this embodiment also yielded a fourth expression box and a fifth expression box using the same construction and transformation method as in Embodiment 1:

[0133] Fourth expression box:

[0134] US_308a / TPI1p-ERG12-GND2t / TEF2p-ERG8-ENO2t / TEF1p-ERG19-CYC1t / US_308a;

[0135] Fifth Expression Box:

[0136] US_YPRCδ15 / TDH3p-PcPT07-TEF1t / TEF2p-ERG20 F96W / N127W -ENO2t / DS_YP RCδ15.

[0137] The aforementioned expression cassette and repair template were transformed into *Saccharomyces cerevisiae* BY4742 (whose genome already contained a pre-integrated Cas9 gene expression cassette) along with the corresponding sgRNA plasmids (sgRNA-911b / 1014a / 416d / PDC5 / ARO10 / 308a / YPRCδ5). After colony PCR verification of correct integration, *Saccharomyces cerevisiae* BAK03, capable of de novo synthesis of bakuchiol, was obtained.

[0138] The engineered strain BAK03 was cultured overnight in YPD medium at 30°C, and then inoculated into 10 mL of YPD medium at OD = 0.2. Under the same conditions, it was cultured for three days. After the culture was complete, 0.5 mL of the bacterial culture was taken, sonicated, and extracted three times with an equal volume of ethyl acetate. The organic phases were combined, dried under vacuum, and reconstituted with 200 μL of methanol. After filtration through a 0.22 μm filter, liquid chromatography analysis was performed. The results showed that the engineered strain BAK03 of *Saccharomyces cerevisiae* could produce p-coumaric acid and psoralen, with a titer of 13.2 mg / L for p-coumaric acid and 78.4 μg / L for psoralen.

[0139] Example 3

[0140] In order to increase the yield of bakuchiol, this embodiment, based on Example 2, constructed a brewer's yeast engineered strain BAK04-07 that can stably produce high yields of bakuchiol.

[0141] Using the *Saccharomyces cerevisiae* BY4742 genome as a template, the 807-base sequence preceding the squalene epoxidase ERG1 ATG was expanded by PCR to obtain the pERG1 promoter fragment. Using the *Saccharomyces cerevisiae* BY4742 genome as a template, the sequence beyond 290 bp upstream of the ERG20 start codon ATG was expanded by PCR as the upstream homologous arm (US_ERG20), and the ERG20 gene sequence was amplified as the downstream homologous arm (DS_ERG20). These fragments were assembled by fusion PCR and ligated into the pTA2 vector via TA cloning. After sequencing confirmed the integrated fragments, the corresponding gene expression cassette, US_ERG20-ERG1p-DS_ERG20, was obtained.

[0142] The supplied DNA and the corresponding sgRNA plasmid (sgRNA-ERG20) were co-transformed into the engineered Saccharomyces cerevisiae strain BAK03. After colony PCR verification confirmed correct integration, the engineered Saccharomyces cerevisiae strain BAK04 was obtained.

[0143] The fermentation method and sample processing method were the same as in Example 2. Analysis of the fermentation results showed that BAK04 could produce 236.85 μg / L of psoralen, which is twice the yield of BAK03.

[0144] To further increase the yield of psoralen, this embodiment incorporates ERG20. F96W / N127W The proteins ERG20 and PcPT07 spatially approach the substrate, thereby improving catalytic efficiency. ERG20 is then used in fusion PCR with a linker (GGGGS) to... F96W / N127WThe fusion gene fragment was obtained by fusing the fragment to the N-terminus of PcPT07. This fragment was then assembled with the corresponding promoter and terminator via fusion PCR and ligated into the pTA2 vector using TA cloning. After successful sequencing, the corresponding gene expression cassette was obtained.

[0145] PGK1p-ERG20 F96W / N127W -GGGGS-PcPT07t-TEF2t;

[0146] FBA1p-ERG20 F96W / N127W -GGGGS-PcPT07t-VPS13;

[0147] ENO2p-ERG20 F96W / N127W -GGGGS-PcPT07t-CYC1t.

[0148] Based on the above gene expression cassettes, single-copy, double-copy, and triple-copy expression cassettes were constructed, as follows:

[0149] Single-copy expression box:

[0150] US_XII-2 / PGK1p-ERG20 F96W / N127W -GGGGS-PcPT07t-TEF2t / DS_XII-2;

[0151] Double-copy expression box:

[0152] US_XII-2 / PGK1p-ERG20 F96W / N127W -GGGGS-PcPT07t-TEF2t / FBA1p-ERG20F96W / N127W-GGGGS-PcPT07t-VPS13 / DS_XII-2;

[0153] Three-copy expression box:

[0154] US_XII-2 / PGK1p-ERG20 F96W / N127W -GGGGS-PcPT07t-TEF2t / FBA1p-ERG20 F96W / N127W -GGGGS-PcPT07t-VPS13 / ENO2p-ERG20 F96W / N127W -GGGGS-PcPT07t-CYC 1t / DS_XII-2.

[0155] The single-copy expression cassette and sgRNA plasmid (sgRNA-XII-2) were co-transformed into Saccharomyces cerevisiae BAK04. After colony PCR verification that the integration was correct, the engineered Saccharomyces cerevisiae strain BAK05 was obtained.

[0156] In addition, the double-copy expression cassette and sgRNA plasmid (sgRNA-XII-2) were co-transformed into Saccharomyces cerevisiae BAK04. After colony PCR verification that the integration was correct, the engineered Saccharomyces cerevisiae strain BAK06 was obtained.

[0157] In addition, the three-copy expression cassette and sgRNA plasmid (sgRNA-XII-2) were co-transformed into Saccharomyces cerevisiae BAK04. After colony PCR verification that the integration was correct, the engineered Saccharomyces cerevisiae strain BAK07 was obtained.

[0158] Single clones of engineered strains BAK05, BAK06, and BAK07 were picked from the plate and placed in 5 mL of YPD medium. Activation was performed by incubation at 30°C and 220 rpm for 20 h. Subsequently, according to OD... 600 =0.2% was re-inoculated into 10 mL of YPD medium. Incubated at 30℃ and 220 rpm for 3 days. After incubation, 500 μL of bacterial culture was taken and 500 μL of methanol was added. The mixture was vortexed and sonicated for 10 min. After sonication, the culture was centrifuged at 14000 rpm for 10 min. The supernatant was filtered through a 0.22 μm filter membrane and then analyzed by liquid chromatography. The results are as follows:

[0159] The amounts of psoralen produced by BAK05, BAK06, and BAK07 were 3.67 mg / L, 7.81 mg / L, and 9.27 mg / L, respectively. Detailed results are shown in Table 1.

[0160] Table 1. Fermentation results of the yeast engineered strains constructed in Examples 2 and 3.

[0161]

[0162] The method for constructing gene expression cassettes and the yeast transformation method in this invention include the following specific steps:

[0163] Preprocessing methods for constructed gene expression cassette fragments

[0164] Pretreatment process for yeast fragment assembly: The total donor DNA fragment volume is 2000 ng (calculate the required volume of each fragment according to its molar ratio). Mix the fragments thoroughly and calculate the total volume to be added. Add 1 / 10 of the total volume of 3M sodium acetate solution. Then add 2 times the total volume of pre-cooled anhydrous ethanol and mix thoroughly. Incubate at -20℃ for 20 min. After incubation, centrifuge at 15000 rpm for 10 min at 4℃ to collect the nucleic acid precipitate. Discard the supernatant and wash the precipitate with 100 μL of 70% ethanol. Centrifuge under the same conditions for 5 min. After centrifugation, discard the supernatant, wash the precipitate with 100 μL of 70% ethanol, and centrifuge again, discarding the supernatant. Finally, dry at room temperature for 10 min and reconstitute the precipitate with 4 μL of ddH2O. Store the solution at -20℃ for later use.

[0165] Chemical transformation methods of brewing yeast

[0166] This invention utilizes the Frozen-EZ Yeast Transformation II Kit from ZYMO Research for yeast transformation. The specific steps are as follows:

[0167] (1) Pick a single yeast colony from the streaked YPD plate and incubate it overnight at 30°C and 220 rpm in 5 mL of YPD medium.

[0168] (2) The next day, take 100 μL of the overnight culture and culture it in 10 mL of YPD medium under the same conditions until OD. 600 =0.8-1.0. Take 500 μL of bacterial culture, centrifuge the cells at 500×g for 4 min, and discard the supernatant;

[0169] (3) Add 500 μL of EZ1 solution to resuspend the cells, centrifuge under the same conditions, and discard the supernatant;

[0170] (4) Add 50 μL of EZ2 solution to resuspend the cells to obtain competent yeast cells;

[0171] (5) Mix 4 μL of donor DNA fragment, 1 μL of sgRNA plasmid (about 500 ng) with 50 μL of yeast competent cells, add 500 μL of EZ3 solution, and mix thoroughly.

[0172] Incubate at 30°C for 60 minutes, gently tapping the solution with your finger every 20 minutes to mix it.

[0173] Spread 200 μL of the solution onto the corresponding amino acid auxotrophic plate. After the plate is dried, incubate it upside down in a 30°C incubator for 4-6 days.

[0174] Integration of Cas9 gene expression cassette

[0175] To facilitate subsequent gene manipulation of *Saccharomyces cerevisiae* using the CRISPR / Cas9 method, this invention integrates the Cas9 gene expression cassette into the HIS3 site of *Saccharomyces cerevisiae* BY4742. The specific steps are as follows: Primers were designed to clone the Cas9 expression cassette (TEF1p-Cas9-CYC1t) from addgene#43802; primers were designed to clone the G418 resistance gene expression cassette (KanMX) from addgene#39296; primers were designed to clone the upstream and downstream homologous arms of HIS3 from the *Saccharomyces cerevisiae* BY4742 genome. Following the aforementioned chemical transformation method, the expression cassette (US_HIS3 / TEF1-Cas9-CYC1t / KanMX / DS_HIS3) was integrated into the *Saccharomyces cerevisiae* BY4742 genome. Positive transformants were screened on G418YPD plates containing 500 μg / mL. Positive transformants were further validated by colony PCR to confirm the presence of the Cas9 expression cassette on the *Saccharomyces cerevisiae* genome.

[0176] The Saccharomyces cerevisiae engineered strains in the above embodiments were all constructed based on Saccharomyces cerevisiae BY4742, which integrates the Cas9 gene expression cassette.

[0177] Preparation method of sgRNA plasmid in the examples

[0178] Based on addgene#43803, the first 20 bases of the gRNA scaffold sequence were replaced by PCR amplification. The corresponding sequences of the integration site are shown in Table 2.

[0179] Table 2 Sequence listing of integration sites

[0180]

[0181] sgRNA plasmid recovery method

[0182] To facilitate subsequent gene manipulation using this plasmid, the sgRNA plasmid was recovered. The yeast strain carrying the sgRNA plasmid was inoculated into 5 mL of YPD medium and cultured at 30°C and 220 rpm for 12 h (the culture time was extended appropriately if the strain grew slowly). After culture, 10 μL of the bacterial culture was transferred to a fresh 5 mL YPD medium and cultured for 12 h under the same conditions. Then, 1 mL of the bacterial culture was centrifuged at 500 × g for 4 min, the supernatant was discarded, and the bacterial pellet was washed with 1 mL ddH2O. This process was repeated, centrifuging again and discarding the supernatant. The bacterial pellet was resuspended in 1 mL ddH2O. 10 μL of this solution was transferred to 1 mL ddH2O. The mixture was thoroughly mixed by pipetting, and 200 μL of the solution was spread onto a YPD plate containing 5-fluoroorotic acid. After the solution dried, the plate was inverted and incubated at 30°C for 4 days. After culturing, single clones were picked and streaked onto YPD plates containing 5-fluoroorotic acid and uracil-deficient synthetic medium plates, respectively. They were incubated upside down at 30°C. A single clone that grows on a YPD plate containing 5-fluoroorotic acid but not on a uracil-deficient synthetic medium plate is considered a successfully recovered sgRNA plasmid strain.

[0183] The culture medium and its components involved in this invention are specifically as follows:

[0184] YPD medium: 1% Yeast Extract, 2% Tryptone, 2% Glucose. If preparing solid plates, add 2% agar powder.

[0185] Uracil-deficient synthesis medium: 1.7 g / L yeast basic nitrogen source (YNB), 5 g / L ammonium sulfate, 20 g / L glucose; the following amino acids are used at 0.1 g / L: arginine, cysteine, lysine, threonine, tryptophan, leucine, adenine; the following amino acids are used at 0.05 g / L: aspartic acid, isoleucine, phenylalanine, proline, serine, tyrosine, valine, methionine, histidine. For solid plates, 15 g / L agar powder should be added.

[0186] The amino acid and nucleotide sequences involved in this invention are as follows:

[0187] SEQ ID NO.1:

[0188] Name: Amino acid sequence of mutant isopentenyltransferase PcPT07

[0189] Sequence type: AA

[0190] Biological origin: synthetic construct

[0191] MAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIITLCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWK RYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYFIIPLMR

[0192] SEQ ID NO.2:

[0193] Name: Nucleotide sequence of the gene encoding the mutant isopentenyltransferase PcPT07

[0194] Sequence type: DNA (other DNA)

[0195] Biological origin: synthetic construct

[0196] ATGGCAGCCAGTGGTGAATCATTAGAATCTCATGAAGCTCAACATCATACTCCTGAAACCTTGTTGGGTTCTATAAAACGTTTTTGTGATGCTTTTTACAGGTTTAGTAGGCCACACGTGATAACTGGCACAGCAATAAACATAATTGTTATGTCTTCCCTTGCATTGGAGAACTCATCAGATATTTCTACGAAATTTTTTATTGGCTTGTTCCAGGCCATCCTAACTACCATTCCTATGAACCTTTACGCTTCAGGCTTGAATCAGTTAACTGACATTGAAATAGACAAGATAAATAAACCGTATCTTCCACTAGCATCGGGGGAGTTTTCTGTTAGAACTGGTGTCATTATTATTACATTGTGTGCGATTATGAGTTTATTGGTTGGATGGGTTGTAGGTTCACCAGCATTATTTTGGACTAGTTTTGCCTATTTTGTGCTAGGGACTGTTTATACAATCAATTTGCCCCTATTGAGATGGAAGAGATATCCAGCGTTTGCAGCTTTGTGCTTCTTCATTATTCGAGGGCTAATGTTTCATGTTGCCTTTTTTCTTCACATACAGACCCATGTGTTCAAGAGGCCAATGATGATTCCAAAATCAGTGATGTTTGGTACGGCATTCATGAGTTTCTTCTATGTGATTATAGCATTGTTCAAGGATATACCTGATATTGAAGGAGATAAAGAACACGGTGTTAAATCTTTGACAATGCGTTTGGGTCAGGAGCGGGTATTCTGGATTTGTGTTTCACTTCTTTTAATGGCTTATGGAGGTGCCATTGTTGTGGGAGCAACATCTTCCTTCCTCTGGAGCAAATTGATCACGGTTTCGGGACATGCTCTTCTAGCTTCAATTTTCTGGAACCGTGCCAACTCTGTTGATTTGAAAAGTCACGAAGAGATAACATCCTTATATATGTTTATGTGGAAGCTATTTTATATTGAATACTTCATAATACCTTTAATGAGATAA

[0197] SEQ ID NO.3:

[0198] Name: Mutant 3-deoxy-D-arabinohepetulose-7-phosphate synthase ARO4 K229L Nucleotide sequence of encoding gene

[0199] Sequence type: DNA (other DNA)

[0200] Biological origin: synthetic construct

[0201]

[0202] SEQ ID NO.4:

[0203] Name: Chorismate mutase ARO7 G141S Nucleotide sequence of the encoding gene

[0204] Sequence type: DNA (other DNA)

[0205] Organism source: synthetic construct

[0206] ATGGATTTCACAAAACCAGAAACTGTTTTAAATCTACAAAATATTAGAGATGAATTAGTTAGAATGGAGGATTCGATCATCTTCAAATTTATTGAGAGGTCGCATTTCGCCACATGTCCTTCAGTTTATGAGGCAAACCATCCAGGTTTAGAAATTCCGAATTTTAAAGGATCTTTCTTGGATTGGGCTCTTTCAAATCTTGAAATTGCGCATTCTCGCATCAGAAGATTCGAATCACCTGATGAAACTCCCTTCTTTCCTGACAAGATTCAGAAATCATTCTTACCGAGCATTAACTACCCACAAATTTTGGCGCCTTATGCCCCAGAAGTTAATTACAATGATAAAATAAAAAAAGTTTATATTGAAAAGATTATACCATTAATTTCGAAAAGAGATGGTGATGATAAGAATAACTTCTCTTCTGTTGCCACTAGAGATATAGAATGTTTGCAAAGCTTGAGTAGGAGAATCCACTTTGGCAAGTTTGTTGCTGAAGCCAAGTTCCAATCGGATATCCCGCTATACACAAAGCTGATCAAAAGTAAAGATGTCGAGGGGATAATGAAGAATATCACCAATTCTGCCGTTGAAGAAAAGATTCTAGAAAGATTAACTAAGAAGGCTGAAGTCTATGGTGTGGACCCTACCAACGAGTCAGGTGAAAGAAGGATTACTCCAGAATATTTGGTAAAAATTTATAAGGAAATTGTTATACCTATCACTAAGGAAGTTGAGGTGGAATACTTGCTAAGAAGGTTGGAAGAGTAA

[0207] SEQ ID NO.5:

[0208] Name: Mutant mevalonate synthase EfMvaS A110G Nucleotide sequence type of encoding genes: DNA (other DNA)

[0209] Biological origin: synthetic construct

[0210]

[0211] SEQ ID NO.6:

[0212] Name: Target sequence of integration site 911b

[0213] Sequence type: DNA (other DNA)

[0214] Biological origin: synthetic construct

[0215] GTAATATTGTCTTGTTTCCC

[0216] SEQ ID NO.7:

[0217] Name: Target sequence of integration site 308a; Sequence type: DNA (other DNA); Organism origin: synthetic construct; SEQ ID NO. 8: CACTTGTCAAACAGAATATA

[0218] Name: Target sequence of integration site YPRCδ5 Sequence type: DNA (other DNA) Organism origin: synthetic construct SEQ ID NO.9: AATCCGAACAACAGAGCATA

[0219] Name: Target sequence of integration site 1014a Sequence type: DNA (other DNA) Organism origin: synthetic construct SEQ ID NO.10: TTATGTGCGTATTGCTTTCA

[0220] Name: Target sequence at integration site 416d; Sequence type: DNA (other DNA); Organism origin: synthetic construct; SEQ ID NO. 11:

[0221] Name: Target sequence of integration site XII-2 Sequence type: DNA (other DNA) Organism origin: synthetic construct SEQ ID NO.12:

[0222] Name: Target sequence of integration site PDC5; Sequence type: DNA (other DNA); Organism origin: synthetic construct; SEQ ID NO. 13:

[0223] Name: Target sequence of integration site ARO10; Sequence type: DNA (other DNA); Organism origin: synthetic construct

[0224] CCTTCAAACAAAATTAATCT

[0225] SEQ ID NO.14:

[0226] Name: Target sequence of integration site ERG20

[0227] Sequence type: DNA (other DNA)

[0228] Biological origin: synthetic construct

[0229] CGAAGTCAGCTTCTTCTCGT

[0230] SEQ ID NO.15:

[0231] Name: Amino acid sequence of isopentenyltransferase PcPT07-FL

[0232] Sequence type: AA

[0233] Biological origin: synthetic construct

[0234] MASMLFLGSLPLASSANYIGRSTRSKKCTESYHATSYITTASWNKTEKIQHEYANMRHRQHNLKHNYGGIEGVSTCEDWARNFVVNAASGESLESHEAQHHTPETLLGSIKRFCDAFYRFSRPHVITGTAINIIVMSSLALENSSDISTKFFIGLFQAILTTIPMNLYASGLNQLTDIEIDKINKPYLPLASGEFSVRTGVIIIT LCAIMSLLVGWVVGSPALFWTSFAYFVLGTVYTINLPLLRWKRYPAFAALCFFIIRGLMFHVAFFLHIQTHVFKRPMMIPKSVMFGTAFMSFFYVIIALFKDIPDIEGDKEHGVKSLTMRLGQERVFWICVSLLLMAYGGAIVVGATSSFLWSKLITVSGHALLASIFWNRANSVDLKSHEEITSLYMFMWKLFYIEYFIIPLMR

[0235] SEQ ID NO.16:

[0236] Name: Fusion gene fragment (geranyl pyrophosphate synthase mutant ERG20) F96W / N127W The nucleotide sequence after fusion with the truncated isopentenyltransferase PcPT07

[0237] Sequence type: DNA (other DNA)

[0238] Biological origin: synthetic construct

[0239]

[0240] SEQ ID NO.17:

[0241] Name: Nucleotide sequence of the gene encoding the linker peptide

[0242] Sequence type: DNA (other DNA)

[0243] Biological origin: synthetic construct

[0244] GGTGGTGGCGGTTCC

[0245] SEQ ID NO.18:

[0246] Name: Amino acid sequence of the linker peptide

[0247] Sequence type: AA

[0248] Biological origin: synthetic construct

[0249] GGGGS.

[0250] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. However, these modifications or substitutions do not cause the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of *Saccharomyces cerevisiae* that produces bakuchiol, characterized in that: The engineered strain of Saccharomyces cerevisiae overexpresses the gene encoding the gerany pyrophosphate synthesis pathway, the gene encoding the coumaric acid synthesis pathway, and the gene encoding the mutant isopentenyltransferase PcPT07. The genes governing the gerany-based pyrophosphate synthesis pathway include the gene encoding acetyl-CoA thiolase / HMG-CoA reductase EfMvaE from Enterococcus faecalis and the mutant mevalonate synthase EfMvaS. A110G The encoding genes, as well as the isopentenyl diphosphate isomerase IDI1 encoding gene, mevalonate phosphate kinase ERG8 encoding gene, mevalonate diphosphate decarboxylase ERG19 encoding gene, mevalonate kinase ERG12 encoding gene, and geranyl pyrophosphate synthase mutant ERG20 from Saccharomyces cerevisiae BY4742. F96W / N127W Encoding genes; The GenBanK number of the EfMvaE encoding gene is KX064239; The EfMvaS A110G The nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; The genes governing the p-coumaric acid synthesis pathway include the AtPAL2 gene encoding phenylalanine ammonia-lyase, the AtC4H gene encoding cinnamic acid-4-hydroxylase, and the AtATR2 gene encoding P450 reductase from Arabidopsis thaliana; the FjTAL gene encoding tyrosine ammonia-lyase from Flavobacterium; and the mutant 3-deoxy-D-arabinohepenolate-7-phosphate synthase ARO4 from Saccharomyces cerevisiae. K229L Encoding gene and cladistic acid mutase ARO7 G141S Encoding genes; The GenBanK number of the AtPAL2 encoding gene is NM_115186.4; The GenBanK number of the AtC4H encoding gene is NM_128601.3; The GenBanK number of the AtATR2 encoding gene is NM_119167.4; The GenBanK number of the FjTAL encoding gene is WP_012023194.1; The ARO4 K229L The nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; The ARO7 G141S The nucleotide sequence of the encoding gene is shown in SEQ ID NO.4; The amino acid sequence of the mutant isopentenyltransferase PcPT07 is shown in SEQ ID NO.

1.

2. The engineered strain of *Saccharomyces cerevisiae* producing bakuchiol according to claim 1, characterized in that: The engineered Saccharomyces cerevisiae strain was introduced into the mutant isopentenyltransferase PcPT07 encoding gene via at least one of the following integration methods: a: The gene encoding the integrated mutant isopentenyltransferase PcPT07; b: Integrate 1-3 fusion gene fragments, wherein the fusion gene fragments are encoded by the mutant isopentenyltransferase PcPT07 gene via a linker peptide encoding gene and the geranyl pyrophosphate synthase mutant ERG20. F96W / N127W It is obtained by linking encoding genes.

3. The engineered strain of *Saccharomyces cerevisiae* producing bakuchiol according to claim 1, characterized in that: The engineered strain of brewer's yeast has also been modified in at least one of the following ways: a) The genes encoding endogenous pyruvate decarboxylase PDC5 and phenylpyruvate decarboxylase ARO10 were knocked out; b) The promoter of the farnesyl pyrophosphate synthase ERG20 gene was replaced in situ with the promoter of the squalene epoxygenase ERG1 gene.

4. The engineered strain of *Saccharomyces cerevisiae* producing bakuchiol according to claim 1, characterized in that: The engineered strain of *Saccharomyces cerevisiae* is classified as *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae BAK07, collection number CGMCCNO.31081.

5. A method for constructing a *Saccharomyces cerevisiae* engineered strain producing bakuchiol as described in any one of claims 1 to 4, characterized in that: The construction method specifically involves: constructing multiple expression cassettes, integrating each expression cassette into the genome of Saccharomyces cerevisiae using homologous recombination technology to obtain a first strain, and using the first strain as the engineered strain; The expression cassette includes a first expression cassette, a second expression cassette, a third expression cassette, a fourth expression cassette, and a fifth expression cassette; the first expression cassette includes the gene encoding phenylalanine ammonia-lyase AtPAL2, the gene encoding cinnamic acid-4-hydroxylase AtC4H, and the gene encoding P450 reductase AtATR2; the second expression cassette includes the gene encoding tyrosine ammonia-lyase FjTAL and the mutant 3-deoxy-D-arabinohepenolate-7-phosphate synthase ARO4. K229L Encoding gene and cladistic acid mutase ARO7 G141S The third expression cassette contains the gene encoding acetyl-CoA thiolase / HMG-CoA reductase EfMvaE and the mutant mevalonate synthase EfMvaS. A110G The fourth expression cassette contains the encoding genes for mevalonate kinase ERG12, mevalonate phosphate kinase ERG8, and mevalonate diphosphate decarboxylase ERG19; the fifth expression cassette contains the geranyl pyrophosphate synthase mutant ERG20. F96W / N127W The encoding gene and the mutant isopentenyltransferase PcPT07 encoding gene.

6. The method for constructing the engineered strain of *Saccharomyces cerevisiae* according to claim 5, characterized in that: The construction method further includes the following steps: The second strain was obtained by knocking out the PDC5 gene encoding endogenous pyruvate decarboxylase and the ARO10 gene encoding phenylpyruvate decarboxylase in the first strain using homologous recombination technology. By using homologous recombination technology, the promoter of the farnesyl pyrophosphate synthase ERG20 gene in the genome of the second strain was replaced in situ with the promoter of the squalene epoxidase ERG1 gene to obtain the third strain. The mutant isopentenyltransferase PcPT07 encoding gene was linked to the geranyl pyrophosphate synthase mutant ERG20 via a linker peptide encoding gene. F96W / N127W The coding genes are linked to obtain a fusion gene fragment; an expression cassette containing 1 to 3 of the fusion gene fragments is integrated into the genome of a third strain using homologous recombination technology to obtain a fourth strain, which is then used as the engineered strain of *Saccharomyces cerevisiae*.

7. The application of a *Saccharomyces cerevisiae* engineered strain producing bakuchiol as described in any one of claims 1 to 4, or a *Saccharomyces cerevisiae* engineered strain constructed using the construction method as described in any one of claims 5 to 6, characterized in that: This method is used to produce psoralen by catalytic trans-geranylation of p-coumaric acid and coupling decarboxylation, using glucose as a substrate and p-coumaric acid as an intermediate.

8. A method for producing bakuchiol using a *Saccharomyces cerevisiae* engineered strain as described in any one of claims 1-4 or a *Saccharomyces cerevisiae* engineered strain constructed using any one of the construction methods as described in claims 5-6, characterized in that: The engineered strain of *Saccharomyces cerevisiae* was cultured in a fermentation medium to obtain a fermentation culture. The fermentation culture was then extracted and separated to obtain psoralen.

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