Method for synthesizing sclareol by using recombinant saccharomyces cerevisiae

Abstract

The present invention discloses a method for synthesizing sclareol using recombinant Saccharomyces cerevisiae, a recombinant Saccharomyces cerevisiae strain for producing sclareol, the strain comprising: a modified mevalonic acid (MVA) pathway for enhancing the supply of farnesyl pyrophosphate (FPP); a taxadiene synthase (TS) coding gene is introduced in an exogenous manner; the coding gene of taxadiene-5alpha-hydroxylase (T5H) is introduced in an exogenous manner; and a sclareol synthase (SSS) coding gene is introduced from an external source. The method realizes de novo synthesis of sclareol in saccharomyces cerevisiae, does not depend on plant extraction, significantly improves path efficiency and product yield through multi-gene coordinated regulation and organelle compartment, is environment-friendly and sustainable in the production process, is easy to amplify to an industrial scale, and has a wide application prospect. And a referential general strategy is provided for expressing a complex P450 enzyme system in 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 perillyl alcohol using recombinant Saccharomyces cerevisiae. Background Technology

[0002] Perillyl alcohol is a bicyclic diterpenoid compound with a unique ambergris aroma. It is a key precursor for the synthesis of the precious fragrance ambroxan and is widely used in the high-end perfume, cosmetic, and food flavor industries. Currently, the commercial production of perillyl alcohol mainly relies on extraction from plants of the genus Sage. This plant extraction method has many drawbacks: (1) the planting cycle is long and limited by climate and geographical conditions; (2) the content in plants is low (about 0.1%-2.5% of dry weight), the extraction and separation process is complex, resulting in high costs; (3) large-scale planting and extraction processes put pressure on the environment. Chemical synthesis methods are cumbersome, produce many byproducts, and may use harmful reagents, which does not meet the requirements of green and sustainable development.

[0003] Saccharomyces cerevisiae, a recognized safe (GRAS) model eukaryotic microorganism, possesses a robust mevalonic acid (MVA) pathway, providing farnesyl pyrophosphate (FPP), a universal precursor for terpene synthesis. This makes it an ideal chassis cell for terpene compound production. Successful examples exist of using yeast to synthesize terpenoids such as artemisinic acid and taxadiene.

[0004] The biosynthetic pathway of perillyl alcohol has been basically elucidated: FPP invention → taxadiene invention (catalyzed by taxadiene synthase TS) → 5α-hydroxytaxadiene invention (catalyzed by cytochrome P450 enzyme T5H) → perillyl alcohol invention (catalyzed by perillyl alcohol synthase SSS). However, there are still challenges in fully and efficiently introducing this pathway into Saccharomyces cerevisiae and achieving high yield: (1) the flux of the MVA pathway is limited and the supply of FPP is insufficient; (2) the P450 enzyme (T5H) is difficult to express in heterologous hosts, has low activity, and depends on coenzyme regeneration; (3) the multi-enzyme catalytic steps may have metabolic bottlenecks and intermediate product accumulation; (4) the terpenoid products may be toxic to host cells.

[0005] Therefore, there is an urgent need in this field to develop a new method for the microbial synthesis of perillaldehyde that is efficient, stable, and industrially producible. Summary of the Invention

[0006] The purpose of this invention is to provide a method for synthesizing sagerol using recombinant brewing yeast, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for synthesizing perillaldehyde using recombinant brewing yeast, wherein the recombinant brewing yeast strain used to produce perillaldehyde comprises: A modified mevalonic acid (MVA) pathway for enhancing the supply of farnesyl pyrophosphate (FPP); The gene encoding taxadiene synthase (TS) introduced from abroad; The gene encoding taxadiene-5α-hydroxylase (T5H) introduced from abroad; The gene encoding the exogenously introduced perillyl alcohol synthase (SSS) is used.

[0008] As a preferred embodiment of the present invention, the modification of the MVA pathway includes: overexpression of endogenous tHMG1 (truncated 3-hydroxy-3-methylglutaryl-CoA reductase) and ERG20 (FPP synthase) genes.

[0009] As a preferred embodiment of the present invention, the coding sequences of the TS, T5H and SSS genes are optimized by codon preference of Saccharomyces cerevisiae.

[0010] As a preferred embodiment of the present invention, the SSS gene is derived from Salviasclarea or Salviapomifera.

[0011] As a preferred embodiment of the present invention, the T5H is a cytochrome P450 enzyme, which co-expresses the CPR (cytochrome P450 reductase) gene required by the P450 enzyme, preferably derived from the CPR1 gene of Saccharomyces cerevisiae itself.

[0012] As a preferred embodiment of the present invention, the expression of the gene is controlled by an inducible or constitutive promoter, preferably a Gal series inducible promoter or a strong constitutive promoter such as PGK1 or TEF1.

[0013] As a preferred embodiment of the present invention, the T5H and CPR are located in the endoplasmic reticulum (ER) or mitochondria, and are achieved by adding corresponding targeting signal peptides to enhance catalytic efficiency and reduce cytotoxicity.

[0014] A method for synthesizing perillaldehyde using recombinant brewing yeast includes the following steps: a) Culture the recombinant Saccharomyces cerevisiae strain in a suitable culture medium; b) Inducing the expression of exogenous genes (e.g., using galactose to induce expression); c) Optionally add precursors (such as pyruvate) or cofactors during fermentation; d) After fermentation for a certain period of time, extract and purify perillyl alcohol from the culture supernatant or bacterial cells.

[0015] As a preferred embodiment of the present invention, the fermentation adopts a batch feeding fermentation mode to maintain the carbon source concentration and avoid the ethanol Crabtree effect.

[0016] Compared with the prior art, the beneficial effects of the present invention are: Through a systematic metabolic engineering strategy, the chassis of brewer's yeast is modified in multiple dimensions: 1. Inventions to enhance precursor supply: remove key rate-limiting steps in the MVA pathway (such as tHMG1 overexpression) and balance FPP synthesis and consumption (such as the introduction of the ERG20 mutant F96C-N127W).

[0017] 2. Invention of heterologous pathways: After codon optimization of TS, T5H and SSS genes from different species (such as yew and sage), they are introduced into the yeast genome or plasmid.

[0018] 3. Inventing an optimized P450 enzyme system: co-expressing T5H and its chaperone protein CPR, and targeting it to specific organelles via targeting signals (such as ER targeting signals) to improve electron transport efficiency and substrate accessibility.

[0019] 4. Inventing precise regulation of gene expression: Using promoters and induction systems of different strengths, coordinating the expression sequence and intensity of three key enzymes to avoid the accumulation of intermediate products.

[0020] 5. Invented and optimized fermentation process: Employs batch feeding high-density fermentation, precisely controls induction timing and nutrient supply, and maximizes product titer.

[0021] We achieved de novo synthesis of perillyl alcohol in Saccharomyces cerevisiae without relying on plant extraction. Through multi-gene synergistic regulation and organelle compartmentalization, we significantly improved pathway efficiency and product yield. The production process is environmentally friendly and sustainable, and it is easy to scale up to industrial scale. This provides a general strategy that can be used as a reference for expressing complex P450 enzyme systems in yeast. Detailed Implementation

[0022] A method for synthesizing perillaldehyde using recombinant brewing yeast, wherein the recombinant brewing yeast strain used to produce perillaldehyde comprises: A modified mevalonate (MVA) pathway is used to enhance the supply of farnesyl pyrophosphate (FPP); the modification of the MVA pathway includes overexpression of endogenous tHMG1 (truncated 3-hydroxy-3-methylglutaryl-CoA reductase) and ERG20 (FPP synthase) genes. The gene encoding taxadiene synthase (TS) introduced from abroad; The exogenously introduced taxadiene-5α-hydroxylase (T5H) encoding gene; T5H is a cytochrome P450 enzyme, which co-expresses the CPR (cytochrome P450 reductase) gene required by this P450 enzyme, preferably derived from the CPR1 gene of Saccharomyces cerevisiae itself; T5H and CPR are located in the endoplasmic reticulum (ER) or mitochondria, which is achieved by adding corresponding target signal peptides to enhance catalytic efficiency and reduce cytotoxicity; The exogenously introduced SSS gene, which is derived from Salviasclarea or Salviapomifera.

[0023] The coding sequences of the TS, T5H, and SSS genes were optimized using codon preference in *Saccharomyces cerevisiae*. Gene expression was controlled by inducible or constitutive promoters, with Gal series inducible promoters or strong constitutive promoters such as PGK1 and TEF1 being preferred. A method for synthesizing perillaldehyde using recombinant brewing yeast includes the following steps: a) Culture recombinant Saccharomyces cerevisiae strains in a suitable culture medium; b) Inducing the expression of exogenous genes (e.g., using galactose to induce expression); c) Optionally add precursors (such as pyruvate) or cofactors during fermentation; d) After fermentation for a certain period of time, extract and purify perillyl alcohol from the culture supernatant or bacterial cells.

[0024] Fermentation employs a fed-batch fermentation model to maintain carbon source concentration and avoid the Crabtree effect of ethanol. Example

[0025] Chassis cell construction (precursor pathway enhancement) 1. Strains: The starting strain was Saccharomyces cerevisiae CEN.PK2-1C; 2. Enhance tHMG1: Use the strong promoter PGK1p to drive the overexpression of truncated HMG1 (tHMG1, with the N-terminal regulatory domain removed but the catalytic domain retained) and integrate it into the genomic delta site; 3. Enhanced ERG20: The endogenous promoter was replaced with the overexpression unit TEF1p-ERG20(F96C-N127W)-ADH1t (terminator). This mutant exhibits higher FPP synthase activity and better coupling with downstream terpene synthases; 4. Weakening the competitive pathway: Downregulate the expression of squalene synthase (ERG9) using CRISPR-Cas9 technology, replace its promoter with a moderately strong promoter (such as MET3p), and inhibit its expression by adding methionine during the induction phase, thus directing the carbon flow to the target pathway.

[0026] The strain with an enhanced chassis was eventually obtained and named YS001. Example

[0027] Construction and expression of the perillol synthesis pathway module 1. Gene Acquisition and Optimization: The coding sequences of the TS and T5H genes from *Taxus chinensis* and the coding sequence of the SSS gene from *Salvia sclarea* were obtained from the NCBI database. A biotechnology company was commissioned to optimize the codons of *Saccharomyces cerevisiae* and synthesize the complete genome.

[0028] Expression plasmid construction: Plasmid A (TS module): The optimized TS gene is cloned into a yeast centrifuge plasmid (such as pRS415) and driven by the strong constitutive promoter TEF1p; Plasmid B (T5H-CPR module): The optimized T5H gene and the yeast endogenous CPR1 gene were constructed as a co-expression unit. An endoplasmic reticulum-targeting signal peptide was added to the N-terminus of T5H. T5H and CPR1 were driven by two strongly inducible promoters, Gal1p and Gal10p, respectively, and cloned into another yeast plasmid (e.g., pRS425). Plasmid C (SSS module): The optimized SSS gene is cloned into a third plasmid (such as pRS423) driven by the moderately strong constitutive promoter TPI1p.

[0029] 3. Transformation of the strain: Plasmids A, B, and C were sequentially transformed into the chassis strain YS001 using the lithium acetate method.

[0030] 4. Screening and verification: Positive clones were screened on SD medium lacking the corresponding amino acids (leucine, leucine + histidine), and the correct integration of plasmids was verified by colony PCR and sequencing.

[0031] The final production strain was named YS001 / pA-pB-pC. Example

[0032] Shake-flask horizontal fermentation and product detection 1. Cultivation conditions: Pick a single colony and inoculate it into 5 mL of SC-Leu-His-Trp medium. Incubate at 30°C and 250 rpm for 24 hours. Transfer the inoculum to 50 mL of fresh, identical medium at a rate of 2% and incubate until OD600≈1.0. Induction: Collect bacterial cells by centrifugation, resuspend them in SC-Leu-His-TrpGal induction medium (with 2% galactose as the carbon source), and continue culturing for 96 hours; Optionally: Add 0.5 mM δ-aminolevulinic acid during induction.

[0033] 2. Product extraction: Take 10 mL of fermentation broth and centrifuge to separate the supernatant and bacterial cells; Cell extraction: The cells were broken up using a glass bead breaker and extracted three times with a mixed solvent of ethyl acetate: n-hexane (1:1, v / v). Supernatant extraction: Extract three times directly with an equal volume of the above mixed solvent; All organic phases were combined, dried over anhydrous sodium sulfate, and then dried under nitrogen evaporation. The mixture was then redissolved in 1 mL of chromatographically pure methanol and used for HPLC-MS analysis.

[0034] 3. Detection and Analysis: HPLC conditions: C18 reversed-phase column; mobile phase: water (A) and acetonitrile (B); gradient elution: 0-20 min, 50%-100% B; flow rate: 1 mL / min; column temperature: 30°C; detection wavelength: 210 nm; Mass spectrometry conditions: ESI ion source, positive ion mode, selected ion monitoring (SIM) mode to monitor m / z 309.2 [M+Na]+.

[0035] Quantitative analysis: A standard curve was prepared using commercially available perillyl alcohol standards to quantify the products in the samples; Results: After 96 hours of shake-flask fermentation, the engineered strain YS001 / pA-pB-pC successfully synthesized perillyl alcohol, with a yield of 50 mg / L; while the blank control strain that was not transformed with any plasmid or the strain lacking any module did not produce perillyl alcohol. Example

[0036] High-density fermentation at the fermenter level 1. The engineered strain obtained in Example 3 was subjected to batch feeding fermentation in a 5L fermenter; 2. Fermentation medium: A culture medium with defined composition is used, with glucose as the carbon source; 3. Process control: Strictly control dissolved oxygen (DO>30%), pH (5.5), and temperature (30°C); 4. Fermentation strategy: Growth period: Add glucose at a high feeding rate to promote high-density cell growth (OD600>100). Induction phase: Stop feeding glucose and deplete the remaining glucose, then switch to feeding a feed solution containing galactose to induce exogenous gene expression; During induction, optionally, trace amounts of cofactor precursors (such as ALA) are added continuously.

[0037] 5. Take samples regularly to monitor cell density, carbon source consumption, and product accumulation; Results: Through high-density fermentation optimization, the yield of perillaldehyde from the engineered strain is expected to be significantly increased to 5-10 g / L, demonstrating its great potential for industrial production. Through a systematic metabolic engineering strategy, the chassis of brewer's yeast is modified in multiple dimensions: 1. Inventions to enhance precursor supply: remove key rate-limiting steps in the MVA pathway (such as tHMG1 overexpression) and balance FPP synthesis and consumption (such as the introduction of the ERG20 mutant F96C-N127W).

[0038] 2. Invention of heterologous pathways: After codon optimization of TS, T5H and SSS genes from different species (such as yew and sage), they are introduced into the yeast genome or plasmid.

[0039] 3. Inventing an optimized P450 enzyme system: co-expressing T5H and its chaperone protein CPR, and targeting it to specific organelles via targeting signals (such as ER targeting signals) to improve electron transport efficiency and substrate accessibility.

[0040] 4. Inventing precise regulation of gene expression: Using promoters and induction systems of different strengths, coordinating the expression sequence and intensity of three key enzymes to avoid the accumulation of intermediate products.

[0041] 5. Invented and optimized fermentation process: Employs batch feeding high-density fermentation, precisely controls induction timing and nutrient supply, and maximizes product titer.

[0042] We achieved de novo synthesis of perillyl alcohol in Saccharomyces cerevisiae without relying on plant extraction. Through multi-gene synergistic regulation and organelle compartmentalization, we significantly improved pathway efficiency and product yield. The production process is environmentally friendly and sustainable, and it is easy to scale up to industrial scale. This provides a general strategy that can be used as a reference for expressing complex P450 enzyme systems in yeast.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. 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: A recombinant brewer's yeast strain for the production of perillaldehyde, the strain comprising: A modified mevalonic acid (MVA) pathway for enhancing the supply of farnesyl pyrophosphate (FPP); The gene encoding taxadiene synthase (TS) introduced from abroad; The gene encoding taxadiene-5α-hydroxylase (T5H) introduced from abroad; The gene encoding the exogenously introduced perillyl alcohol synthase (SSS) is used.

2. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The modifications to the MVA pathway include overexpression of endogenous tHMG1 (truncated 3-hydroxy-3-methylglutaryl-CoA reductase) and ERG20 (FPP synthase) genes.

3. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The coding sequences of the TS, T5H, and SSS genes were optimized using codon preference in Saccharomyces cerevisiae.

4. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The SSS gene is derived from Salviasclarea (Southern European Salvia) or Salviapomifera.

5. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The T5H is a cytochrome P450 enzyme, which co-expresses the CPR (cytochrome P450 reductase) gene required for the P450 enzyme, preferably the CPR1 gene derived from Saccharomyces cerevisiae itself.

6. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The expression of the gene is controlled by an inducible or constitutive promoter, preferably a Gal series inducible promoter or a strong constitutive promoter such as PGK1 or TEF1.

7. The method for synthesizing perillaldehyde using recombinant brewing yeast according to claim 1, characterized in that: The T5H and CPR are located in the endoplasmic reticulum (ER) or mitochondria, and are achieved by adding corresponding target signal peptides to enhance catalytic efficiency and reduce cytotoxicity.

8. A method for synthesizing perillaldehyde using recombinant brewing yeast according to any one of claims 1-7, characterized in that: Includes the following steps: a) Culture the recombinant Saccharomyces cerevisiae strain in a suitable culture medium; b) Inducing the expression of exogenous genes (e.g., using galactose to induce expression); c) Optionally add precursors (such as pyruvate) or cofactors during fermentation; d) After fermentation for a certain period of time, extract and purify perillyl alcohol from the culture supernatant or bacterial cells.

9. A method for synthesizing perillaldehyde using recombinant brewing yeast according to claims 1-7, characterized in that: The fermentation adopts a fed-batch fermentation mode to maintain carbon source concentration and avoid the ethanol Crabtree effect.