Method for synthesizing sclareolide by using recombinant saccharomyces cerevisiae

Abstract

The invention discloses a method for synthesizing sclareolide by using recombinant saccharomyces cerevisiae, which comprises a recombinant saccharomyces cerevisiae strain for producing sclareolide, and the strain comprises a nucleotide sequence of exogenously coded sclareol synthase (SLS), a nucleotide sequence of exogenously coded cytochrome P450 oxidase and a nucleotide sequence of exogenously coded cytochrome P450 reductase (CPR). According to the invention, by enhancing upstream terpenoid precursor supply, a key rate-limiting enzyme gene, such as tHMG1 (deregulated form for catalyzing HMG-CoA reductase) and ERG20 (FPP synthase), in a mehydroxy acid pathway (MVA pathway) is overexpressed by introducing a strong promoter, and a carbon flow is guided to the sclareol synthesis direction; a sclareol synthesis module is introduced and optimized: a plant-derived sclareol synthase (SLS) gene is introduced, and sclareol synthase can generate sclareol through one-step cyclization by taking FPP as a substrate. The gene is subjected to codon optimization so as to adapt to an expression system of yeast.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and metabolic engineering, and in particular to a method for synthesizing perillaldehyde using recombinant Saccharomyces cerevisiae. Background Technology

[0002] Sclareolide is a bicyclic diterpenoid lactone compound with a warm, amber, and ambergris-like aroma, and is an oxidation product of sclareol. It is an extremely valuable fixative and raw material in the perfume, cosmetics, and fragrance industries, and also exhibits certain antibacterial and pharmacological activities. Currently, sclareolide is mainly extracted from the plant Salvia sclarea. However, plant extraction methods have many drawbacks: 1) yields are unstable due to limitations imposed by plant growth cycles, seasons, and climate conditions; 2) the extraction process is complex and costly; 3) over-cultivation and harvesting may put pressure on the ecological environment. Chemical synthesis methods are cumbersome, produce many byproducts, and do not conform to the current trend of "green chemistry."

[0003] Synthetic biology offers revolutionary solutions for the production of these high-value natural products. By designing and constructing artificial biosynthetic pathways, efficient and sustainable production of target compounds can be achieved in microbial cell factories such as *Saccharomyces cerevisiae*. *Saccharomyces cerevisiae*, a recognized safe (GRAS) model eukaryotic microorganism, possesses complete organelles (such as the endoplasmic reticulum and mitochondria), making it ideally suited for the synthesis of eukaryotic natural products like terpenoids. It possesses a robust terpenoid precursor synthesis pathway from acetyl-CoA to methanogenic acid (MVA).

[0004] Although previous studies have attempted to synthesize the precursor perillyl alcohol in yeast, the direct and efficient synthesis of perillyl lactone still faces challenges: 1) insufficient carbon flux in the terpene synthesis pathway; 2) low expression activity and poor stability of cytochrome P450 enzymes (key enzymes responsible for oxidizing perillyl alcohol to perillyl lactone) in yeast, and dependence on specific cytochrome P450 reductases (CPR); 3) cytotoxicity of intermediate and final products; 4) the products may be metabolized or secreted inefficiently by the yeast itself. Therefore, there is an urgent need in this field to develop a recombinant Saccharomyces cerevisiae strain and method that has undergone multiple metabolic engineering modifications to efficiently and stably produce and secrete perillaldehyde. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synthesizing perilla lactone using recombinant brewing yeast, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for synthesizing perillaldehyde using recombinant Saccharomyces cerevisiae, comprising a recombinant Saccharomyces cerevisiae strain for producing perillaldehyde, wherein the strain contains a nucleic acid sequence encoding exogenous perillyl alcohol synthase (SLS), an exogenous nucleic acid sequence encoding cytochrome P450 oxidase, and an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR).

[0007] As a preferred embodiment of the present invention, the cytochrome P450 oxidase is CYP76AH1 or its functional homolog.

[0008] As a preferred embodiment of the present invention, the coding sequences of cytochrome P450 oxidase and CPR are linked by self-cleaving peptides (such as P2A, T2A) or internal ribosome entry sites (IRES) to achieve co-expression; or expressed as a fusion protein.

[0009] As a preferred embodiment of the present invention, one or more genes of the endogenous methanogenic pathway (MVA) are further enhanced in expression, including tHMG1 and ERG20.

[0010] As a preferred embodiment of the present invention, the expression of the endogenous squalene synthase gene ERG9 is further weakened or knocked out.

[0011] As a preferred embodiment of the present invention, the efflux transporter gene PDR5 or SNQ2 is further overexpressed.

[0012] As a preferred embodiment of the present invention, a method for producing perilla lactone includes the following steps: a) Using the method of synthesizing perilla lactone from recombinant Saccharomyces cerevisiae, the recombinant Saccharomyces cerevisiae strain as described in any one of 1-6 is cultured in a fermentation medium; b) Recovering perillaldehyde from cultures or culture media by using recombinant Saccharomyces cerevisiae to synthesize perillaldehyde.

[0013] As a preferred embodiment of the present invention, the fermentation adopts a fed-batch fermentation mode, and galactose or other inducers are used to induce the expression of P450 and CPR genes.

[0014] As a preferred embodiment of the present invention, the method for synthesizing perillaldehyde using recombinant brewing yeast yields perillaldehyde higher than 0.5 g / L.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention systematically modifies the Saccharomyces cerevisiae host through the following multi-level metabolic engineering strategies: a) Enhance the supply of upstream terpene precursors: By introducing strong promoters to overexpress key rate-limiting enzyme genes in the methanogenic pathway (MVA pathway), such as tHMG1 (the deregulated form of HMG-CoA reductase) and ERG20 (FPP synthase), carbon flow is directed toward the synthesis of perillaldehyde; b) Introduction and optimization of the perillaldehyde synthesis module: A plant-derived perillaldehyde synthase (SLS) gene was introduced, which can cyclize FPP as a substrate to generate perillaldehyde in one step. The gene was codon-optimized to suit the yeast expression system; c) Introduction and optimization of the perillaldehyde oxidation module: This is the core of the invention. We introduce a highly active cytochrome P450 enzyme (CYP76AH1 or a functional homolog) responsible for the stepwise oxidation of perillaldehyde to perillaldehyde, and finally to perillic acid, while simultaneously introducing its optimally matched cytochrome P450 reductase (CPR1). To further improve oxidation efficiency, we construct a fusion protein of the P450 enzyme and CPR, or co-anchor them to the mitochondrial membrane to enhance electron transport and substrate channel effects. d) Introducing a lactone cyclization step: Introducing a lactone cyclase capable of catalyzing the spontaneous or enzymatic cyclization of perillic acid to form perillyl lactone; in some embodiments, spontaneous cyclization under acidic conditions is sufficient.

[0016] e) Enhance product secretion and tolerance: Overexpress efflux transport proteins (such as PDR5, SNQ2) or modify cell wall permeability to reduce product feedback inhibition and cytotoxicity.

[0017] f) Weakening the competition pathway: Knocking out or downregulating genes that compete with farnesyl pyrophosphate (FPP) using technologies such as CRISPR-Cas9, such as the promoter of ERG9 (squalene synthase), directs carbon flow to the target product. Detailed Implementation

[0018] A method for synthesizing perillaldehyde using recombinant Saccharomyces cerevisiae includes a recombinant Saccharomyces cerevisiae strain for producing perillaldehyde, the strain containing a nucleic acid sequence encoding exogenous perillyl alcohol synthase (SLS), an exogenous nucleic acid sequence encoding cytochrome P450 oxidase, and an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR), wherein the cytochrome P450 oxidase is CYP76AH1 or a functional homolog thereof, and the coding sequences of cytochrome P450 oxidase and CPR are linked by self-cleaving peptides (such as P2A, T2A) or internal ribosome entry sites (IRES) to achieve co-expression; or expressed as a fusion protein.

[0019] Further enhancement of expression of one or more genes in the endogenous methanogenic pathway (MVA), including tHMG1 and ERG20; Further overexpression of efflux transporter genes PDR5 or SNQ2; A method for producing perilla lactone, comprising the steps of: a) Using the method of synthesizing perilla lactone from recombinant Saccharomyces cerevisiae, a recombinant Saccharomyces cerevisiae strain is cultured in a fermentation medium according to any one of claims 1-6; b) Recovering perillaldehyde from cultures or culture media by using recombinant Saccharomyces cerevisiae to synthesize perillaldehyde.

[0020] Fermentation was carried out using a fed-batch fermentation model, and galactose or other inducers were used to induce the expression of the P450 and CPR genes. The yield of perillaldehyde was higher than 0.5 g / L using a method for synthesizing perillaldehyde using recombinant Saccharomyces cerevisiae. Example

[0021] Construction of basic engineered strains Host strain: Saccharomyces cerevisiae CEN.PK2-1C was selected as the initial host; Enhance the MVA pathway: Design primers to amplify the tHMG1 (Δ1-552aa) coding sequence with the degradation domain removed from the yeast genome; The tHMG1 fragment was cloned into the multicopy yeast expression vector pRS425, which has a strong promoter (such as pGPD) and a strong terminator (such as tCYC1), to construct the plasmid pRS425-GPD-tHMG1-TCYC1. Similarly, the coding sequence of the endogenous ERG20 gene was cloned into the expression vector pRS426-GPD to construct pRS426-GPD-ERG20-TCYC1.

[0022] Introducing the perillol synthesis module: The codons of the Sacchariol synthase (SLS) gene from Salviasclarea were optimized in Sacchari yeast, and the whole gene was synthesized. The optimized SLS gene was cloned into the expression vector pRS423-TEF1p to construct the plasmid pRS423-TEF1-SLS-TCYC1. Introducing the perillol oxidation module: Select CYP76AH1 from Coleusblumei (known to oxidize diterpenoid-like compounds) and its matching CPR1 gene; The coding sequences of CYP76AH1 and CPR1 were linked together using the DNA sequence of the P2A "self-cleaving" peptide chain to form an open reading frame. This construct was cloned into the pRS416-GAL1p (galactose-inducible promoter) vector to construct the plasmid pRS416-GAL1-CYP76AH1-P2A-CPR1-TCYC1. This was intended to achieve co-expression of the two proteins.

[0023] Transformation and strain construction: All the above plasmids (pRS425-tHMG1, pRS426-ERG20, pRS423-SLS, pRS416-CYP-CPR) were co-transformed into Saccharomyces cerevisiae CEN.PK2-1C competent cells using the lithium acetic acid method. Transformants were screened on synthetic complete (SC) media plates lacking the corresponding amino acids (leucine, uracil, histidine) to obtain the basic engineered strain Sc-SL01; Example

[0024] Construction of advanced engineered strains (integration and knockout) Ways to weaken competition: We designed a CRISPR-Cas9 system to downregulate the expression of ERG9 (squalene synthase), designed gRNA to target the promoter region of ERG9, and co-transformed it with the Cas9 expression cassette into strain Sc-SL01. Simultaneously, a homologous recombination repair template containing a weak promoter (such as pENO2) of ERG9 is provided to replace its original strong promoter, thereby reducing but not completely eliminating the expression of ERG9 and maintaining the cell growth required.

[0025] Stable expression through genome integration: The expression cassettes of tHMG1 and SLS were integrated into the δ site (stable region) of the yeast genome via homologous recombination. The fusion-expressed *CYP76AH1-P2A-CPR1* unit was integrated into another neutral site; Ultimately, a plasmid-independent, genetically stable advanced engineered strain, Sc-SL02, was obtained. Example

[0026] Fermentation production of perilla lactone Seed culture preparation: Pick a single colony Sc-SL02 from the plate and inoculate it into 5 mL of SC-Leu-His-Ura liquid medium. Incubate at 30°C and 250 rpm for 24 hours.

[0027] Batch fermentation: The seed culture was transferred at a 1:50 inoculum to a 2L fermenter containing 500mL of fermentation medium (YPD or synthetic medium with appropriate trace elements). Fermentation conditions: 30°C, pH maintained at 5.5, dissolved oxygen (DO) maintained above 30% by adjusting the stirring speed.

[0028] Induction and Feeding: When OD600 reached ~15 (mid-logarithmic growth), galactose was added to a final concentration of 2% (w / v) to induce GAL1 promoter-driven CYP / CPR expression. Simultaneously, feedmed medium containing glucose was added to maintain a low glucose concentration (~5 g / L) to avoid the Crabtree effect.

[0029] Product extraction and detection: After 96 hours of fermentation, the fermentation broth was collected.

[0030] Extraction: The supernatant and bacterial cells were separated by centrifugation. The supernatant was extracted twice with an equal volume of ethyl acetate. The bacterial cells were disrupted using a glass bead disruptor and then extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation.

[0031] Detection: The concentrate was analyzed by gas chromatography-mass spectrometry (GC-MS). Qualitative analysis was performed by comparing retention time and characteristic ion fragments (m / z) with that of styracil lactone standard, and quantification was performed by external standard method.

[0032] Results: After 96 hours of fermentation, the engineered strain Sc-SL02 produced 1.2 g / L of perillaldehyde, with approximately 80% of the product secreted into the fermentation supernatant, greatly facilitating downstream separation and purification. Perillaldehyde was not detected in the control strain (containing only the empty vector).

[0033] 1. Construction strategy of engineered strains: This invention systematically modifies the Saccharomyces cerevisiae host through the following multi-level metabolic engineering strategies: a) Enhance the supply of upstream terpene precursors: By introducing strong promoters to overexpress key rate-limiting enzyme genes in the methanogenic pathway (MVA pathway), such as tHMG1 (the deregulated form of HMG-CoA reductase) and ERG20 (FPP synthase), carbon flow is directed toward the synthesis of perillaldehyde; b) Introduction and optimization of the perillaldehyde synthesis module: A plant-derived perillaldehyde synthase (SLS) gene was introduced, which can cyclize FPP as a substrate to generate perillaldehyde in one step. The gene was codon-optimized to suit the yeast expression system; c) Introduction and optimization of the perillaldehyde oxidation module: This is the core of the invention. We introduce a highly active cytochrome P450 enzyme (CYP76AH1 or a functional homolog) responsible for the stepwise oxidation of perillaldehyde to perillaldehyde, and finally to perillic acid, while simultaneously introducing its optimally matched cytochrome P450 reductase (CPR1). To further improve oxidation efficiency, we construct a fusion protein of the P450 enzyme and CPR, or co-anchor them to the mitochondrial membrane to enhance electron transport and substrate channel effects. d) Introducing a lactone cyclization step: Introducing a lactone cyclase capable of catalyzing the spontaneous or enzymatic cyclization of perillic acid to form perillyl lactone; in some embodiments, spontaneous cyclization under acidic conditions is sufficient.

[0034] e) Enhance product secretion and tolerance: Overexpress efflux transport proteins (such as PDR5, SNQ2) or modify cell wall permeability to reduce product feedback inhibition and cytotoxicity.

[0035] f) Weakening the competition pathway: Knocking out or downregulating genes that compete with farnesyl pyrophosphate (FPP) using technologies such as CRISPR-Cas9, such as the promoter of ERG9 (squalene synthase), directs carbon flow to the target product.

[0036] 2. Recombinant Saccharomyces cerevisiae strain: The strain contains expression cassettes of the following exogenous genes: Sage alcohol synthase (SLS) gene Cytochrome P450 oxidase (CYP) gene Cytochrome P450 reductase (CPR) gene lactone cyclase gene Furthermore, the expression of key genes in the endogenous MVA pathway was enhanced, while the expression of competing pathways was weakened.

[0037] 3. Production method: The above-mentioned engineered strains were cultured in a suitable fermentation medium. Carbon source concentration, pH and dissolved oxygen were controlled by fed-batch fermentation mode. Finally, perilla lactone was isolated and purified from the fermentation supernatant or cell extract.

[0038] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for synthesizing perillaldehyde using recombinant brewing yeast, characterized in that: This includes a recombinant Saccharomyces cerevisiae strain that produces perillaldehyde, the strain containing a nucleic acid sequence encoding exogenous perillaldehyde synthase (SLS), an exogenous nucleic acid sequence encoding cytochrome P450 oxidase, and an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR).

2. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The cytochrome P450 oxidase is CYP76AH1 or its functional homolog.

3. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The coding sequences for cytochrome P450 oxidase and CPR are linked via self-cleaving peptides (such as P2A and T2A) or internal ribosome entry sites (IRES) to achieve co-expression; or expressed as a fusion protein.

4. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: Further enhancement expression of one or more genes in the endogenous methanogenic pathway (MVA), including tHMG1 and ERG20.

5. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: Further, the expression of the endogenous squalene synthase gene ERG9 was weakened or knocked out.

6. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: Further overexpression of efflux transporter genes PDR5 or SNQ2.

7. A method for synthesizing perillaldehyde using recombinant brewing yeast according to any one of claims 1-6, characterized in that... ： A method for producing perilla lactone, comprising the steps of: a) Using the method of synthesizing perilla lactone from recombinant Saccharomyces cerevisiae, the recombinant Saccharomyces cerevisiae strain as described in any one of 1-6 is cultured in a fermentation medium; b) Recovering perillaldehyde from cultures or culture media by using recombinant Saccharomyces cerevisiae to synthesize perillaldehyde.

8. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 7, characterized in that: The fermentation adopts a fed-batch fermentation mode, and galactose or other inducers are used to induce the expression of P450 and CPR genes.

9. A method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 7, characterized in that: The method for synthesizing perillaldehyde using recombinant brewer's yeast yields a perillaldehyde yield higher than 0.5 g / L.