Sclareol synthase variants and engineered yeast producing sclareol

Abstract

The present application belongs to the field of biosynthesis, and particularly relates to a sclareol synthase variant and a yeast engineering bacterium for producing sclareol. In order to solve the problem of generally low yield of sclareol biosynthesis in the prior art, the present application mutates and optimizes the sclareol synthase, obtains a SsScs mutant containing a G307L mutation, and uses the mutant for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability existing in the expression of free plasmid in some Saccharomyces cerevisiae strains, the present application constructs a sclareol synthesis pathway in the yeast genome, simultaneously strengthens the acetyl coenzyme A synthesis pathway, the MVA pathway and the sclareol synthesis pathway, and knocks out part of the transcriptional repressor to transform the chassis bacterium for synthesizing sclareol, and the yield is greatly improved compared with the prior art.

Description

[0001] This application is a divisional application of invention patent application 202610010720.9, which was filed on January 6, 2026, with application number 202610010720.9, and the invention title is: Perillyl alcohol synthase and engineered yeast strain for producing perillyl alcohol. Technical Field

[0003] This invention belongs to the field of biosynthesis, specifically relating to a variant of perillaldehyde synthase and an engineered yeast strain for producing perillaldehyde. Background Technology

[0005] In 2012, Swedish researchers (Schalk M, Pastore L, Mirata MA, et al. Towarda biosynthetic route to sclareol and amber odorants.[J]. Journal of the American Chemical Society, 2012, 134(46):18900-3.) discovered two key enzymes for the synthesis of sclareol in Salvia sclarea, namely lysabenaenoyl pyrophosphate synthase (SsLPPs3) and sclareol synthase (SsTPs1132). These enzymes catalyze the reactions of geranyl geranyl pyrophosphate (GGPP) to lysabenaenoyl pyrophosphate (LPP) and LPP to sclareol, respectively. Using Escherichia coli as a host, they heterologously expressed the MVA pathway and the sclareol biosynthetic pathway, combined with high-density fermentation technology, and synthesized sclareol in a two-phase culture with dodecane as the organic phase, achieving a yield of 1.5 g / L.

[0006] Einhaus et al. (Einhaus A, Steube J, Freudenberg RA, et al. Engineering apowerful green cell factory for robust photoautotrophic diterpenoid production [J]. Metabolic Engineering, 2022, 73: 82-90) overcame the bottleneck of the MEP pathway in Chlamydomonas reinhardtii through enzyme fusion and overexpressed the MEP pathway gene to promote heterologous diterpenoid production. Finally, on day 19, they obtained 656 mg / L of styraxol through high-density photoautotrophic fermentation in a 2.5 L photobioreactor.

[0007] Yang et al. (Yang W, Zhou Y, Liu W, et al. Engineering Saccharomycescerevisiae for sclareol production [J]. Chinese Journal of Biotechnology, 2013, 29(8): 1185-1192) constructed a heterologous biosynthetic pathway of sclareol using Saccharomyces cerevisiae as the host and expressing LPPS and TPS (encoding SsLPS and SsScS, respectively). By overexpressing the key precursor metabolic enzyme tHmg1 and enhancing the substrate channel effect through protein fusion, the engineered strain S6 obtained by combination optimization achieved a sclareol yield of 8.96 mg / L under shake flask conditions. Based on this, the research group constructed a diploid yeast strain S7 (Song Y, Shen H, Yang W, et al. Highcell density culture of an engineered yeast strain for sclareol production[J]. Chinese Journal of Biotechnology, 2015, 31(1): 147-151), and carried out high-density culture in a 3 L fermenter using a feed-dissolved oxygen linkage control method with a glucose / ethanol mixture as the carbon source. The highest yield of sclareol reached 408 mg / L.

[0008] To increase sclareol production, Trikka et al. (Trikka FA, Nikolaidis A, Athanasakoglou A, et al. Iterative carotenogenic screens identify combinations of yeast genedeletions that enhance sclareol production [J]. Microbial Cell Factories, 2015, 14(1): 1-19) used iterative screening of carotenoid gene deletion mutants to obtain a Saccharomyces cerevisiae strain with six endogenous gene deletions. By expressing the genes of the sclareol synthesis pathway through free plasmids, the sclareol production was increased by 12 times, and the shake flask yield reached 750 mg / L.

[0009] However, expression in free plasmids is unstable. In a recent study in 2024, Sun et al. (Sun ML, HanY, Yu X, et al. Constructing a green oleaginous yeast cell factory for sustainable production of the plant-derived diterpenoid sclareol [J]. GreenChemistry, 2024, 26: 5202-5210) heterologously expressed the encoding genes of SsLpps and SsScs in Yersinia lipolytica to construct the sclareol synthesis pathway. By overexpressing the rate-limiting enzyme genes tHMG1 and ERG20, truncating the signal peptide of SsLpps, heterologously expressing SsGGPPS and PaGGPPS to enhance GGPP accumulation, and using short peptide tags to construct multi-enzyme complexes to shorten the physical distance between substrate and enzyme (tSsLpps-RIDD / SsScs-RIAD), the shake-flask yield of sclareol increased from 6.02 mg / L to 817.65 mg / L. The yield of sagerol in a fed-batch fermenter reached 12.9 g / L. This is the highest yield of sagerol reported in existing literature for microbial production.

[0010] Although existing technologies have achieved the biosynthesis of perillyl alcohol through different host and metabolic engineering strategies, the yield is generally low; some Saccharomyces cerevisiae strains express it using free plasmids, which has the problem of genetic instability. Summary of the Invention

[0012] To address the aforementioned technical problems, this invention first mutates and optimizes perillyl alcohol synthase (hereinafter referred to as SsSCS). Secondly, it modifies the basal bacteria that synthesize perillyl alcohol by enhancing the synthesis pathways of acetyl-CoA, MVA, and GGPP, and knocking out some transcriptional regulatory factors. Finally, it introduces the SsSCS mutant into the aforementioned basal bacteria, resulting in a significant increase in perillyl alcohol production.

[0013] To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0014] One of the technical solutions provided by this invention is a perilla frutescens alcohol synthase mutant, wherein the mutant is any one of the following (1) or (2):

[0015] (1) It is obtained by at least one mutation of Y491F, G307L or N269F on the basis of wild-type SsSCS from Salvia sclarea as shown in SEQ ID NO.1;

[0016] (2) is a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the mutant amino acid sequence described in (1), and having perilla syrup synthase activity.

[0017] Furthermore, the SsSCS mutant is an SsSCS(Y491F) mutant obtained by mutating the Y491F mutation on the wild-type SsSCS shown in SEQ ID NO.1, and the amino acid sequence of the SsSCS(Y491F) mutant is shown in SEQ ID NO.2;

[0018] Furthermore, the SsSCS mutant is an SsSCS(G307L) mutant obtained by mutating the G307L mutation on the wild-type SsSCS shown in SEQ ID NO.1, and the amino acid sequence of the SsSCS(G307L) mutant is shown in SEQ ID NO.3;

[0019] Furthermore, the SsSCS mutant is an SsSCS(N269F) mutant obtained by mutating the N269F mutation on the wild-type SsSCS shown in SEQ ID NO.1, and the amino acid sequence of the SsSCS(N269F) mutant is shown in SEQ ID NO.4;

[0020] Furthermore, the SsSCS mutant is an SsSCS (Y491F / G307L) mutant obtained by Y491F and G307L mutations on the basis of the wild-type SsSCS shown in SEQ ID NO.1;

[0021] Furthermore, the SsSCS mutant is an SsSCS (Y491F / N269F) mutant obtained by Y491F and N269F mutations on the basis of the wild-type SsSCS shown in SEQ ID NO.1;

[0022] Furthermore, the SsSCS mutant is an SsSCS (Y491F / G307L / N269F) mutant obtained by Y491F, G307L and N269F mutations on the basis of the wild-type SsSCS shown in SEQ ID NO.1.

[0023] The present invention also provides the encoding gene of the SsSCS mutant as described in one of the technical solutions.

[0024] The second technical solution provided by the present invention is the application of the SsSCS mutant described in the first technical solution, especially in the production of perillaldehyde, and more particularly in the catalytic production of perillaldehyde from lysine pyrophosphate (LPP).

[0025] The third technical solution provided by the present invention is a recombinant vector or recombinant strain containing the coding gene of the SsSCS mutant described in the first technical solution;

[0026] Furthermore, the expression plasmids used in the recombinant vector include, but are not limited to: pYES2, pESC URA, pESC-His, etc.

[0027] Furthermore, the hosts used for the recombinant strains include, but are not limited to: Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yersinia lipolytica, Pichia pastoris, Aspergillus nidulans, Streptomyces, etc.

[0028] Furthermore, the host is *Saccharomyces cerevisiae*;

[0029] Preferably, the brewing yeast includes, but is not limited to, brewing yeast (S. cerevisiae) CEN.PK2-1C.

[0030] The fourth technical solution provided by this invention is an engineered bacterium for producing perillaldehyde. Based on the expression of the SsSCS mutant described in one of the technical solutions in a host, the engineered bacterium also undergoes gene editing for any one or more of the following:

[0031] (1) Overexpress at least one of the genes encoding alcohol dehydrogenase ADH2, acetaldehyde dehydrogenase ALD6, and acetyl-CoA synthase ACS to enhance the acetyl-CoA synthesis pathway.

[0032] (2) Overexpress at least one of the genes encoding HMG-CoA reductase tHMG1, isopentenyl diphosphate isomerase IDI, and farnesyl pyrophosphate synthase Erg20 to enhance the MVA pathway;

[0033] (3) Introduce geraniol pyrophosphate synthase GGPPS (CrtE) to enhance GGPP synthesis flux; introduce lysine pyrophosphate diol ester synthase SsLPPS to enhance the perillol synthesis pathway.

[0034] (4) Knock out at least one of the transcriptional repressor factors GAL80, ROX1, or DOS2 to achieve metabolic flux redirection and enhanced product synthesis;

[0035] Furthermore, the host used for the engineered bacteria is Saccharomyces cerevisiae, Yersinia lipolytica, or Pichia pastoris, with Saccharomyces cerevisiae CEN.PK2-1C being the preferred choice.

[0036] Furthermore, the mutants of the SsSCS are expressed in plasmid form or through genome integration;

[0037] More preferably, when the SsSCS mutant is integrated into the genome, the integration site is position 208a of the genome.

[0038] The fifth technical solution provided by the present invention is the application of the engineered bacteria described in the fourth technical solution in the production of perillaldehyde. The application includes, but is not limited to, the process of fermenting and culturing the engineered bacteria and obtaining perillaldehyde from the fermentation products.

[0039] Beneficial effects:

[0040] (1) In order to solve the problem that the biosynthesis yield of perillaldehyde in the prior art is generally low, this invention mutates and optimizes perillaldehyde synthase to obtain an SsSCS mutant containing at least one mutation of Y491F, G307L or N269F. The mutant was used for perillaldehyde synthesis. The results showed that when SsSCS has a single mutation at Y491F, G307L or N269F, the yield increased by 10.40%, 16.49% and 20.22% respectively compared with the unmutated state. On this basis, further combination mutations of double / triple mutations can gradually increase the perillaldehyde yield. When the Y491F, G307L and N269F mutations occur at the same time, the yield increase is the most significant, which is 2.13 times that of the control group.

[0041] (2) To address the technical problem of genetic instability in the expression of some Saccharomyces cerevisiae strains using free plasmids, this invention constructs a perillaldehyde synthesis pathway in the yeast genome. Simultaneously, it modifies the substrate bacteria that synthesize perillaldehyde by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillaldehyde synthesis pathway, and by knocking out some transcriptional repressors. The yield in a 5 L fermenter reaches 21.13 g / L. This yield is higher than the highest level recorded in current technology. Detailed Implementation

[0043] The present invention will now be described through specific embodiments. All technical means not specifically described herein are methods well-known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the present invention.

[0044] In this invention, wild-type perillyl alcohol synthase from Salvia sclarea is mutated and optimized to obtain the SsSCS mutant containing at least one mutation of Y491F, G307L, or N269F. Simultaneously, the basal bacteria synthesizing perillyl alcohol are modified by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillyl alcohol synthesis pathway, and by knocking out some transcriptional repressors. Finally, the SsSCS mutant is introduced into the aforementioned basal bacteria, resulting in a significant increase in perillyl alcohol production.

[0045] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0046] (1) Use the recognized IUPAC nomenclature for amino acid residues, in three-letter / single-letter code form. DNA nucleic acid sequences use the recognized IUPAC nomenclature.

[0047] (2) Identification (naming) principles of SsSCS mutants

[0048] The mutated amino acid in the SsSCS mutant is represented by "original amino acid residue + residue position + substituted amino acid residue". For example, Y491F indicates that the amino acid at position 491 is replaced by phenylalanine (F) from the wild-type SsSCS, and the position number corresponds to the amino acid sequence number of the wild-type SsSCS in SEQ ID NO.1. Information is shown in Table 1 below:

[0049] Table 1

[0050]

[0051] This invention screens for amino acid mutations in SsSCS and found that mutations at the Y491F, G307L, or N269F positions are beneficial for increasing the yield of perillyl alcohol.

[0052] Therefore, proteins that, while retaining the amino acid mutations at positions Y491F, G307L, or N269F, exhibit a certain degree of homology with the mutants in Table 1 by mutating other amino acid sites, such as having 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% homology (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9% homology) and having the same or similar activities as the mutant proteins in Table 1, also fall within the scope of protection of this invention.

[0053] Homology refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences.

[0054] The proteins that have a certain homology with the mutant proteins in Table 1 can be obtained by further conserved amino acid substitution or replacement based on the above mutations. Conserved amino acid substitution or replacement is well known in the art, and those skilled in the art can also perform conserved substitution of amino acids according to the amino acid substitution rules known in the prior art.

[0055] In this invention, the wild-type SsSCS is derived from Salvia sclarea; the amino acid sequence is shown in SEQ ID NO.1:

[0056] MSLAFNVGVTPFSGQRVGSRKEKFPVQGFPVTTPNRSRLIVNCSLTTIDFMAKMKENFKREDDKFPTTTTLRSEDIPSNLCIIDTLQRLGVDQFFQYEINTILDNTFRLWQEKHKVIYGNVTTHAMAFRLLRVKGYEVSSEEL APYGNQEAVSQQTNDLPMIIELYRAANERIYEEERSLEKILAWTTIFLNKQVQDNSIPDKKLHKLVEFYLRNYKGITIRLGARRNLELYDMTYYQALKSTNRFSNLCNEDFLVFAKQDFDIHEAQNQKGLQQLQRWYADCRLDT LNFGRDVVIIANYLASLIIGDHAFDYVRLAFAKTSVLVTIMDDFFDCHGSSQECDKIIELVKEWKENPDAEYGSEELEILFMALYNTVNELAERARVEQGRSVKEFLVKLWVEILSAFKIELDTWSNGTQQSFDEYISSSWLSN GSRLTGLLTMQFVGVKLSDEMLMSEECTDLARHVCMVGRLLNDVCSSEREREENIAGKSYSILLATEKDGRKVSEDEAIAAEINEMVEYHWRKVLQIVYKKESILPRRCKDVFLEMAKGTFYAYGINDELTSPQQSKEDMKSFVF

[0057] 2. All raw materials and reagents involved in the experiments of this invention can be purchased commercially.

[0058] Chassis cells: Saccharomyces cerevisiae CEN.PK2-1C. All strains used in this application were constructed and modified using Saccharomyces cerevisiae CEN.PK2-1C (from the China Microbial Culture Collection, No. Bio-116324).

[0059] The plasmid pScURA was purchased from Shanghai Qincheng Biotechnology Co., Ltd. pScURA is a yeast plasmid backbone with URA3 as the selection marker, whose flexibility makes it an ideal vector for constructing CRISPR systems, biosensors, and genome integration tools.

[0060] Plasmid pIYC04 was purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0061] Plasmid pSPGM1 was purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0062] The plasmid pESC URA27 was constructed using plasmid pESC URA as a template. pESC URA was purchased from Shanghai Jihe Biotechnology Co., Ltd. The construction process of plasmid pESC URA27 is as follows:

[0063] Using PESC URA plasmid as a template and PESG-GAL2-7-F2 and PESG-GAL2-7-R2 as primers, the vector fragment was obtained by PCR amplification; using PESC... Using the URA plasmid as a template, PESG-GAL2-7-F1 and pESC-GAL2-7-R primer pairs, and PESG-GAL2-7-F and pESC-GAL2-7-R1 primer pairs, the multiple cloning site fragment was obtained by PCR amplification. Using the Saccharomyces cerevisiae genome as a template, the GAL2 and GAL7 promoter fragments were obtained by PCR amplification using pGAL2-F and pGAL2-R, and pGAL7-F and pGAL7-R primer pairs, respectively. Using the Saccharomyces cerevisiae genome as a template, the TPS1 and TPGK1 terminator fragments were obtained by PCR amplification using TTPS1-F and TTPS1-R, and TPGK1-F and TPGK1-R primer pairs, respectively. The fragments obtained by the above PCR amplification were then ligated to the vector using homologous recombinase to obtain the plasmid pESC URA27 (see Table 3 for primer details).

[0064] 3. Information on some of the culture media used in this application:

[0065] 1. Composition of SC-URA plate medium: glucose 20 g / L, agar powder 15 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, histidine 0.02 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L.

[0066] 2. Composition of SC plate medium containing 5-FOA (5-fluoroorotic acid): glucose 20 g / L, 5-fluoroorotic acid 1 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, histidine 0.02 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L, uracil 0.02 g / L, agar powder 15 g / L.

[0067] 3. YPD plate culture medium composition: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agar powder 15 g / L.

[0068] 4. SC-HIS-URA medium: glucose 20 g / L, yeast amino acid-free nitrogen source (YNB) 6.7 g / L, leucine 0.1 g / L, tryptophan 0.02 g / L, agar powder 15 g / L.

[0069] 4. The sequence information of some genes or enzymes involved in this application is shown in Table 2.

[0070] Table 2

[0071]

[0072] 5. Some primer information involved in the embodiments of this application is shown in Table 3.

[0073] Table 3

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] The present invention will be further explained and illustrated below through specific embodiments.

[0082] Example 1: Chassis Microbial Optimization

[0083] I. Overexpression of ADH2, ALD6, and ACS (enhancing the acetyl-CoA synthesis pathway)

[0084] Using Saccharomyces cerevisiae CEN.PK2-1C as the starting chassis cell, strain PKC001 was obtained by overexpressing ADH2, ALD6, and ACS. The specific construction steps are as follows:

[0085] The target gene was integrated into the genome of Saccharomyces cerevisiae using the GTR-CRISPR-Cas9 gene editing system. A plasmid was constructed that simultaneously carries a gene expressing the Cas9 protein and an sgRNA sequence targeting the corresponding site, which was used for targeted integration or knockout of the gene.

[0086] 1. Design plasmid pCas9-1622b-308a

[0087] The guide RNA sequences for the 1622b and 308a sites on the genome of *Saccharomyces cerevisiae* CEN.PK2-1C were predicted and designed using CRISPOR (http: / / crispor.tefor.net / ). Highly efficient gRNA sequences without off-target effects were selected. Primers containing dual gRNA sequences (1622b-D-F1 and 308a-D-F2, R1-D, D-F2, DR, D-R2, see Table 3) were designed. PCR amplification was performed using plasmid pScURA as a template to obtain a gene fragment containing gRNAs at the 1622b and 308a sites and the selection marker Ura. This PCR amplified gene fragment was then ligated to the pCas9 plasmid using homologous recombinase to obtain plasmid pCas9-1622b-308a.

[0088] 2. Design Donor DNA

[0089] (1) Using the Saccharomyces cerevisiae genome as a template, ADH2-F and ADH2-R primers were used to amplify the ADH2 coding gene by PCR; using the Saccharomyces cerevisiae genome as a template, ALD6-F and ALD6-R primers were used to amplify the ALD6 coding gene by PCR; using the pESC URA plasmid as a template, GAL1-F and GAL10-R primers were used to amplify the PGAL1-PGAL10 promoter fragment by PCR; using the pESC URA plasmid as a template, TCYC1-R and 1622b-donor-F primers were used to amplify the TCYC1 terminator by PCR, and TADH1 terminator was amplified by PCR using TADH-F and 1622b-donor-R primers, and the upstream and downstream homologous arms (up and down fragments) of the 1622b site were obtained simultaneously; the above fragments were ligated by OE-PCR to obtain the Donor DNA fragment 1: up-TCYC1-ADH2-PGAL1-PGAL10-ALD6-TADH1-down;

[0090] (2) Using the Saccharomyces cerevisiae genome as a template, and ACS2-F and ACS2-R as primers, the ACS coding gene was amplified by PCR. Using pESC URA27 plasmid as a template, and GAL7-R and 308a-donor-F-27 as primers, the PGAL7-PGAL2 promoter-TPGK1 terminator fragment was amplified. Using TTPS1-F and 308a-donor-R-27 as primers, the TTPS1 terminator was amplified. The upstream and downstream homologous arms (up and down fragments) of the 308a site were obtained simultaneously. The above fragments were ligated by OE-PCR to obtain Donor DNA fragment 2: up-TPGK1-PGAL2-PGAL7-ACS-TTPS1-down.

[0091] 3. Donor DNA fragments 1 and 2 obtained in steps 1 and 2 were co-transformed with pCas9-1622b-308a plasmid into S. cerevisiae CEN.PK2-1C competent cells via electroporation. Homologous recombination within the S. cerevisiae was used to integrate the target gene expression cassette. The transformed cells were plated onto SC-URA plates and incubated at 30°C for 2-3 days. Positive clones were screened by colony PCR. Single colonies verified by PCR were re-streaked onto SC plates containing 5-FOA (5-fluoroorotic acid) and incubated at 30°C for 2-3 days to screen for single colonies lacking the Cas9 plasmid. Single clones growing on SC plates containing 5-FOA (5-fluoroorotic acid) were simultaneously streaked onto YPD and SC-URA plates and incubated at 30°C. The bacteria that grew on YPD plates but not on SC-URA plates were identified as gene-edited bacteria. Single clones were selected and incubated on YPD liquid medium at 30°C for 24 h, followed by preservation of the bacterial strain (20% glycerol) to obtain the chassis strain PKC001, which was then preserved at -80°C for later use (20% glycerol). The validation primers were V-1622b-F / R and V-308a-F / R.

[0092] II. Overexpression of tHMG1, IDI, and Erg20 (enhancing the MVA pathway)

[0093] 1. Design the knockout plasmid pCas9-his3b-911b and donor DNA using the same method as steps 1-3 in Part 1. Only the corresponding primers or templates need to be replaced (primer sequences are shown in Table 3).

[0094] The insertion sites for the tHMG1 and IDI encoding genes are the his3b sites in the genome, and the donor DNA is up-TCYC1-tHMG1-PGAL1-PGAL10-IDI-TADH1-down.

[0095] The Erg20 encoding gene was inserted at site 911b of the genome, and the donor DNA was up-TPGK1-PGAL2-PGAL7-ERG20-TTPS1-down.

[0096] 2. The knockout plasmid pCas9-his3b-911b and donor DNA: up-TCYC1-tHMG1-PGAL1-PGAL10-IDI-TADH1-down and up-TPGK1-PGAL2-PGAL7-ERG20-TTPS1-down were co-transformed into the PKC001 strain prepared in Part 1 to obtain the perillaldehyde-synthesizing strain PKC002. The strain was preserved for later use (20% glycerol). The verification primers V-his3b-F / R and V-911b-F / R were used.

[0097] III. Knockout of GAL80, ROX1, and DOS2 (knockout of negative transcriptional regulators)

[0098] The gRNA sequence for the GAL80 site was designed using the same method as in step 1 of Part 1. Using pScURA as a template and gal80-gRNA-F and gRNA1-R as primers, a gene fragment containing the gRNA for the GAL80 site and the selection marker Ura was obtained. Using the pCas9 plasmid as a template and Cas9-gRNA-F and Cas9-gRNA-R as primers, the pCas9 vector backbone was obtained. The gene fragment obtained by the above PCR amplification was then ligated to the pCas9 vector backbone using homologous recombinase to obtain the plasmid pCas9-GAL80. Without a template, the knockout donor DNA with the upstream and downstream homologous arms (up and down fragments) of the GAL80 site was obtained directly by PCR reaction using primers gal80-ko-donor-F and gal80-ko-donor-R: up-GAL80-knockout-down.

[0099] The knockout plasmid pCas9-GAL80 and donor DNA:up-GAL80-knockout-down were co-transformed into the PKC002 strain prepared in the second part to obtain the PKC003 strain that successfully knocked out the GAL80 gene and synthesized perillyl alcohol. The strain was then stored for later use (20% glycerol).

[0100] 2. Design the knockout plasmid pCas9-ROX1-DOS2 and donor DNA using the same method as in step 1: up-ROX1-knockout-down and up-DOS2-knockout-down. Only the corresponding primers or templates need to be replaced (primer sequences are shown in Table 3).

[0101] The knockout plasmid pCas9-ROX1-DOS2 and donor DNA: up-ROX1-knockout-down and up-DOS2-knockout-down were co-transformed into the PKC003 strain prepared in step 1 to obtain the perillaldehyde synthesizing strain PKC004, which successfully knocked out the ROX1 and DOS2 genes. The strain was then stored for later use (20% glycerol).

[0102] IV. Introducing gerany-gerany pyrophosphate synthase (CrtE) from Pharfia redis to enhance GGPP synthesis flux.

[0103] 1. Design the knockout plasmid pCas9-106a using the same method as steps 1-3 in Part 1 (using pScURA as a template, 106a-gRNA-F and gRNA1-R as primers to obtain a gene fragment containing gRNA at the 106a site and the selection marker Ura; using the pCas9 plasmid as a template, Cas9-gRNA-F and Cas9-gRNA-R as primers to obtain the pCas9 vector backbone; then using homologous recombinase to ligate the gene fragment obtained by the above PCR amplification to the pCas9 vector backbone to obtain plasmid pCas9-106a) and donor DNA: up-TCYC1-PGAL1-PGAL10-CrtE-TADH1-down, only replacing the corresponding primers or template (primer sequences are shown in Table 3). The insertion site for the GGPPS encoding gene is at position 106a in the genome.

[0104] 2. The knockout plasmid pCas9-106a and donor DNA: up-TCYC1-PGAL1-PGAL10-CrtE-TADH1-down were co-transformed into the PKC004 strain prepared in Part III to obtain the perillaldehyde-synthesizing strain PKC005. The strain was preserved for later use (20% glycerol). The primer V-106a-F / R was validated.

[0105] Example 2 Construction of SsLPPS expression plasmid

[0106] The SsLPPS encoding gene was codon-optimized for Saccharomyces cerevisiae (the optimized nucleic acid sequence is shown in SEQ ID NO. 5), and the full-length DNA sequence was synthesized.

[0107] I. Construction of pIYC04-SsLPPS plasmid

[0108] Using pIYC04 as the expression vector, recombinant expression plasmids were constructed using the In-Fusion strategy.

[0109] Using the DNA sequence synthesized according to SEQ ID NO.5 as a template, the SsLPPS gene fragment was amplified by PCR using LPPS-F and LPPS-R primers; using the pIYC04 vector as a template, the linearized pIYC04 vector was amplified by PCR using TADH-F and PTEF-R primers. Following the kit instructions, the obtained SsLPPS gene fragment and the linearized pIYC04 vector fragment were subjected to homologous recombination. After incubation at 50℃ for 1 h, the cells were transformed into E. coli DH5α competent cells, plated on Amp-resistant plates, and incubated overnight at 37℃. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB liquid medium containing Amp for sequencing verification. The correctly sequenced recombinant plasmid pIYC04-SsLPPS was extracted for later use.

[0110] Example 3 Construction of SsSCS mutant plasmid

[0111] The SsSCS encoding gene was codon-optimized for Saccharomyces cerevisiae (the optimized nucleic acid sequence is shown in SEQ ID NO. 6), and the full-length DNA sequence was synthesized.

[0112] Using pSPGM1 as the expression vector, recombinant expression plasmids were constructed using the In-Fusion strategy.

[0113] Using the DNA sequence synthesized according to SEQ ID NO.6 as a template, and SCS-F and SCS-R as primers, the SsSCS gene fragment was amplified by PCR. Using the pSPGM1 vector as a template, and TADH-F and PTEF-R as primers, the linearized pSPGM1 vector was amplified by PCR. Following the kit instructions, the obtained SsSCS gene fragment and the linearized pSPGM1 vector fragment were subjected to homologous recombination. After incubation at 50°C for 1 hour, the cells were transformed into E. coli DH5α competent cells, plated on Amp-resistant plates, and incubated overnight at 37°C. Positive clones were screened by colony PCR, and each positive clone was picked and placed in 5 mL of LB broth containing Amp for sequencing verification. The correctly sequenced recombinant plasmid pSPGM1-SsSCS was extracted for later use.

[0114] II. Construction of SsSCS mutant plasmid

[0115] Homology modeling of SsSCS was performed using AlphaFold3, and then the substrate LPP was docked to the active site of the SsSCS protein using the molecular docking software AutoDock. By analyzing the amino acid residues and steric hindrance of the receptor-ligand interaction, Y491F, G307L or N269F were identified as the key amino acid residues to be mutated.

[0116] 5. Using pSPGM1-SsSCS as a template, mutant primers (SCS-Y491F-F and LPPS-Y491F-R) were designed based on the Y491F single-point mutation for PCR amplification. The PCR product was digested with restriction endonuclease DpnI for 2 hours to remove the template. After agarose gel electrophoresis, the target band was recovered and eluted with ddH2O. The recovered band was then transformed into E. coli DH5α competent cells, plated on Amp-resistant plates, and cultured overnight at 37°C. Positive clones were screened by colony PCR. Positive clones were picked and placed in 5 mL of LB liquid medium containing Amp for sequencing verification. The correct plasmid pSPGM1-SsSCS (Y491F) was extracted for later use.

[0117] 6. The construction methods for the remaining plasmids are the same as for the Y491F single-point mutation. The corresponding mutation primers were replaced according to the different mutation sites (primers are shown in Table 3), resulting in the following plasmids:

[0118] 7 pSPGM1-SsSCS (Y491F);

[0119] 8 pSPGM1-SsSCS (G307L);

[0120] 9 pSPGM1-SsSCS (N269F);

[0121] 10 pSPGM1-SsSCS (Y491F, G307L);

[0122] 11 pSPGM1-SsSCS (Y491F, N269F);

[0123] 12 pSPGM1-SsSCS (Y491F, G307L, N269F).

[0124] Example 4 Construction of recombinant strains (SsLPPS, SsSCS / mutant plasmid expression)

[0125] As shown in Table 4, the different plasmids constructed in Examples 2 and 3 were co-transformed into strain PKC005 (at this time, GGPPS, SsLPPS, and SsSCS or their mutants constitute a complete perillaldehyde synthesis pathway) to obtain recombinant strains CSS001-CSS006 and control strain CON-CS01, which were frozen at -80°C for later use. Specific information is shown in Table 4 below.

[0126] Table 4

[0127]

[0128] Example 5: Perillyl alcohol production experiment (SsLPPS, SsSCS / mutant plasmid expression)

[0129] Shake-flask fermentation:

[0130] Frozen strains CSS001-CSS006 and control strain CON-CS01 were taken from a -80℃ freezer and streaked on SC-HIS-URA plates, then cultured at 30℃ for 2-3 days. Single colonies were picked from the plates and cultured in 10 mL of SC-HIS-URA medium overnight at 30℃ and 250 rpm. The seed culture was transferred at a 5% inoculum to a 250 mL shake flask containing 25 mL of SC-HIS-URA liquid medium and fermented at 30ºC and 250 rpm, with three replicates per group. After 24 h of fermentation, 10% of the fermentation volume of dodecane solvent was added for biphasic fermentation. After 120 h of fermentation, the organic phase was collected by centrifugation at 12,000 rpm for 10 min, diluted with ethanol, and analyzed by HPLC. The specific results are shown in Table 5.

[0131] Table 5

[0132]

[0133] As shown in Table 5, the perillaldehyde yield of the recombinant control strain CON-CS01 expressing wild-type SsLPPS and SsSCS after 120 h of fermentation was 434.33 mg / L, while the perillaldehyde yield of the recombinant strains CSS001-CSS006 expressing SsSCS mutant and wild-type SsLPPS after 120 h of fermentation reached 479.47 mg / L-927.20 mg / L, representing an increase of 10.40%-113% compared to the control group. Specifically, when SsSCS underwent single mutations at positions Y491F, G307L, or N269F, the yield increased by 10.40%, 16.49%, and 20.22%, respectively. Further double / triple mutations gradually increased perillaldehyde yield, with the most significant increase (2.13 times that of the control group) occurring when all three mutations were present.

[0134] Example 6: Construction of Recombinant Strains (SsLPPS, SsSCS / Mutant Genome Integration)

[0135] To further improve the genetic stability of the recombinant strains, SsLPPS and SsSCS or their mutants were integrated into the genome.

[0136] 1. Design the knockout plasmid pCas9-208a using the same method as steps 1-3 in Part 1 (using pScURA as a template, 208a-gRNA-F and gRNA1-R as primers to obtain a gene fragment containing gRNA at the 208a site and the selection marker Ura; using the pCas9 plasmid as a template, Cas9-gRNA-F and Cas9-gRNA-R as primers to obtain the pCas9 vector backbone; then using homologous recombinase to ligate the gene fragment obtained by the above PCR amplification to the pCas9 vector backbone to obtain plasmid pCas9-208a) and donor DNA: up-TCYC1-SsLPPS-PGAL1-PGAL10-SsSCS-TADH1-down, only replacing the corresponding primers or templates (primers are shown in Table 3).

[0137] The insertion site for the coding genes of SsLPPS and SsSCS or their mutants is the 208a site of the genome, and the donor DNA is up-TCYC1-SsLPPS-PGAL1-PGAL10-SsSCS / mutant-TADH1-down.

[0138] 2. The knockout plasmid pCas9-208a and donor DNA: up-TCYC1-SsLPPS-PGAL1-PGAL10-SsSCS / mutant-TADH1-down were co-transformed into strain PKC005 to obtain the perillaldehyde synthesizing strains CSS007-CSS012 (see Table 6 for details) and the control strain CON-CS02. The strains were stored for later use (20% glycerol).

[0139] Table 6

[0140]

[0141] Example 7: Perillyl alcohol production experiment (SsLPPS, SsSCS / mutant genome integration)

[0142] 1. Shake-flask fermentation:

[0143] The frozen strains CSS007-CSS012 and the control strain CON-CS02 were taken out of the -80℃ freezer and fermented in shake flasks using the same method as in Example 5. After fermentation for 120 h, the organic phase was collected by centrifugation at 12,000 rpm for 10 min. After diluting with ethanol by the corresponding factor, styracil alcohol was detected by HPLC. The specific results are shown in Table 7.

[0144] Table 7

[0145]

[0146] As can be seen from the data in Table 7, the yield of genome-integrated SsLPPS and SsSCS / mutant genes is significantly higher than that of plasmid expression in Table 5, but the overall trend remains the same.

[0147] Furthermore, the perillaldehyde yield of the recombinant control strain CON-CS02 expressing wild-type SsSCS and SsSCS reached 567.48 mg / L after 120 h of fermentation, while the perillaldehyde yield of the recombinant strain CSS007-CSS012 expressing SsSCS mutant and wild-type SsLPPS reached 621.92 mg / L-1237.25 mg / L after 120 h of fermentation, representing an increase of 9.59%-118.03% compared to the control group. Specifically, when SsSCS underwent a single mutation at positions Y491F, G307L, or N269F, the yield increased by 9.59%, 12.98%, and 14.22%, respectively. Further double / triple mutations gradually increased perillaldehyde yield, with the most significant increase (2.2 times that of the control group) occurring when all three mutations were present.

[0148] 2.5L fermentation tank:

[0149] The SsSCS tri-strain CSS012, which yielded the highest fermentation yield in shake flasks, was removed from the -80℃ freezer, streaked on YPD plates, and incubated at 30℃ for 2-3 days. Single colonies were picked from the plates and cultured overnight at 30℃ and 250 rpm. The culture was then transferred to a 5L fermenter at a 10% inoculum. Fermentation was controlled at pH 5.0, temperature 30℃, dissolved oxygen (DO) at 30%–40%, with the fermentation speed and dissolved oxygen levels adjusted in tandem. Aeration was maintained at 1 vvm and pressure at 0.02 MPa.

[0150] Prepare the feeding medium according to Table 8, and start feeding after 10 hours of fermentation: monitor the residual sugar and ethanol concentrations of the fermentation broth in real time, and dynamically add the medium (prepared according to Table 8) at a flow rate of 0.1-3 ml / h to keep the residual sugar below 2 g / L and the ethanol below 5 g / L.

[0151] After 24 hours of fermentation, 10% (v / v) dodecane solvent was added for biphase fermentation.

[0152] The composition of the fermentation medium, fed medium, and trace elements is shown in Table 8 below.

[0153] Table 8

[0154]

[0155] The fermentation cycle was 120 h. Samples were taken to test the yield of perillaldehyde. The yields of the three parallel experiments were 21.25, 20.98 and 21.25 g / L, respectively, with an average yield of 21.13 g / L.

[0156] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A perilla frutescens alcohol synthase mutant, characterized in that, The mutant is: The amino acid sequence of the perilla alcohol synthase mutant obtained by the G307L mutation on the basis of the wild-type perilla alcohol synthase shown in SEQ ID NO.1 is shown in SEQ ID NO.

3.

2. The application of the perillaldehyde synthase mutant of claim 1 in the production of perillaldehyde, or in the catalytic production of perillaldehyde from lysine pyrophosphate.

3. The encoding gene of the perilla ethanol synthase mutant according to claim 1.

4. A recombinant vector or recombinant strain containing the encoding gene of claim 3.

5. The recombinant vector or recombinant strain as described in claim 4, characterized in that, The recombinant vectors used expression plasmids including: pYES2, pESC URA, and pESC-His; The hosts used for the recombinant strains include: Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Yersinia lipolytica, Pichia pastoris, Aspergillus nidulans, and Streptomyces.

6. An engineered bacterium for producing perillaldehyde, characterized in that, Based on the expression of the perilla frutescens alcohol synthase mutant of claim 1 in the host, the engineered bacteria also undergo gene editing of any one or more of the following: (1) Overexpressing at least one of the genes encoding alcohol dehydrogenase ADH2, acetaldehyde dehydrogenase ALD6, and acetyl-CoA synthase ACS; (2) Overexpression of at least one of the genes encoding HMG-CoA reductase tHMG1, isopentenyl diphosphate isomerase IDI, and farnesyl pyrophosphate synthase Erg20; (3) Introduce geraniol pyrophosphate synthase GGPPS; Introduce lysine pyrophosphate diol ester synthase SsLPPS; (4) Knock out at least one of the transcriptional repressor factors GAL80, ROX1, or DOS2.

7. The engineered bacteria for producing perillaldehyde as described in claim 6, characterized in that, The engineered bacteria are hosted by Saccharomyces cerevisiae, Yersinia lipolytica, or Pichia pastoris.

8. The use of the engineered bacteria described in claim 6 or 7 in the production of perillaldehyde.

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