Yeast engineering for the production of sclerol by lysophosphatidic acid synthase

Abstract

The present application belongs to the field of biosynthesis, and particularly relates to pyrophosphate lyddane diol ester synthase and yeast engineering bacteria for producing sclareol. In order to solve the problem of generally low yield of sclareol biosynthesis in the prior art, the pyrophosphate lyddane diol ester synthase is mutated and optimized in the present application, and a SsLPPs mutant containing at least one mutation of D730L, D374L, N674L or G379M is obtained, and the mutant is used for sclareol synthesis. Meanwhile, in order to solve the problem of genetic instability existing in the expression of free plasmid in part of saccharomyces cerevisiae strains, the present application constructs a sclareol synthesis pathway in the yeast genome, and at the same time, the acetyl coenzyme A synthesis pathway, the MVA pathway and the sclareol synthesis pathway are strengthened, and part of the transcriptional repressor is knocked out, so that the bottom bacteria for synthesizing sclareol are modified. The yield in a 5 L fermenter reaches 26.11 g / L, and the yield is greatly improved compared with the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis, specifically relating to lysine pyrophosphate diol synthase and engineered yeast strains for producing perillyl alcohol. Background Technology

[0002] 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.) conducted a study on *Salvia miltiorrhiza* (Salvia miltiorrhiza) in Southern Europe. Salvia sclarea Two key enzymes for the synthesis of perillaldehyde were discovered: lysine pyrophosphate diol synthase (SsLPPs3) and perillaldehyde synthase (SsTPs1132). These enzymes catalyze the reactions of gerany gerany pyrophosphate (GGPP) to lysine pyrophosphate diol (LPP) and LPP to perillaldehyde, respectively. Using Escherichia coli as a host, the MVA pathway and the perillaldehyde biosynthetic pathway were heterologously expressed. Combined with high-density fermentation technology, perillaldehyde was synthesized in a two-phase culture with dodecane as the organic phase, achieving a yield of 1.5 g / L.

[0003] 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) reported on Chlamydomonas reinhardtii (… Chlamydomonas reinhardtii In this study, the bottleneck of the MEP pathway was overcome by enzyme fusion, and the MEP pathway gene was overexpressed to promote the heterologous production of diterpenes. Finally, on day 19, 656 mg / L of perillol was obtained by photoautotrophic high-density fermentation in a 2.5 L photobioreactor.

[0004] 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.

[0005] 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.

[0006] 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 perillaldehyde 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 perillaldehyde 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.

[0007] 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

[0008] To address the aforementioned technical problems, this invention first mutates and optimizes lysine pyrophosphate diol ester synthase (hereinafter referred to as SsLPPs). Secondly, it modifies the basal bacteria that synthesize perillyl by enhancing the synthesis pathways of acetyl-CoA, MVA, and GGPP, and knocking out some transcriptional regulatory factors. Finally, it introduces the SsLPPs mutant into the aforementioned basal bacteria, resulting in a significant increase in perillyl yield.

[0009] To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0010] One of the technical solutions provided by this invention is a mutant of lysine pyrophosphate diol ester synthase, wherein the mutant is any one of the following (1) or (2):

[0011] (1) is the fragrant perilla shown in SEQ ID NO.1 ( Salvia sclarea It is obtained by at least one mutation of D730L, D374L, N674L or G379M on wild-type SsLPPs of origin.

[0012] (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 of (1), and having lysine pyrophosphate diol ester synthase activity.

[0013] Furthermore, the SsLPPs mutant is the SsLPPs(D730L) mutant obtained by mutating the D730L mutation on the wild-type SsLPPs shown in SEQ ID NO.1, and the amino acid sequence of the SsLPPs(D730L) mutant is shown in SEQ ID NO.2;

[0014] Furthermore, the SsLPPs mutant is the SsLPPs(D374L) mutant obtained by D374L mutation on the basis of wild-type SsLPPs shown in SEQ ID NO.1, and the amino acid sequence of the SsLPPs(D374L) mutant is shown in SEQ ID NO.3;

[0015] Furthermore, the SsLPPs mutant is the SsLPPs(N674L) mutant obtained by the N674L mutation on the wild-type SsLPPs shown in SEQ ID NO.1, and the amino acid sequence of the SsLPPs(N674L) mutant is shown in SEQ ID NO.4;

[0016] Furthermore, the SsLPPs mutant is an SsLPPs (G379M) mutant obtained by mutating the G379M mutation on the wild-type SsLPPs shown in SEQ ID NO.1, and the amino acid sequence of the SsLPPs (G379M) mutant is shown in SEQ ID NO.5;

[0017] Furthermore, the SsLPPs mutant is an SsLPPs (D730L / D374L) mutant obtained by D730L and D374L mutations on the basis of wild-type SsLPPs shown in SEQ ID NO.1;

[0018] Furthermore, the SsLPPs mutant is an SsLPPs (D730L / N674L) mutant obtained by D730L and N674L mutations on the basis of wild-type SsLPPs shown in SEQ ID NO.1;

[0019] Furthermore, the SsLPPs mutant is an SsLPPs (D730L / G379M) mutant obtained by D730L and G379M mutations on the basis of wild-type SsLPPs shown in SEQ ID NO.1;

[0020] Furthermore, the SsLPPs mutant is an SsLPPs (D730L / D374L / N674L) mutant obtained by D730L, D374L and N674L mutations on the basis of wild-type SsLPPs shown in SEQ ID NO.1;

[0021] Furthermore, the SsLPPs mutant is an SsLPPs (D730L / D374L / G379M) mutant obtained by D730L, D374L and G379M mutations on the basis of wild-type SsLPPs shown in SEQ ID NO.1;

[0022] Furthermore, the SsLPPs mutant is an SsLPPs (D730L / D374L / N674L / G379M) mutant obtained by D730L, D374L, N674L and G379M mutations on the basis of wild-type SsLPPs shown in SEQ ID NO.1.

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

[0024] The second technical solution provided by this invention is the application of the SsLPPs mutant described in the first technical solution, particularly in the production of geraniol, and more particularly in the catalytic production of lysine pyrophosphate from geraniol.

[0025] The third technical solution provided by the present invention is a recombinant vector or recombinant strain containing the coding gene of the SsLPPs 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 SsLPPs 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 perillyl alcohol synthase SsSCS to enhance perillyl alcohol 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 SsLPPS are expressed in plasmid form or through genome integration;

[0037] More preferably, when the SsLPPS 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) To address the generally low yield of perillaldehyde in existing technologies, this invention mutates and optimizes lysine diol pyrophosphate synthase to obtain SsLPPs mutants containing at least one mutation of D730L, D374L, N674L, or G379M. These mutants were then used for perillaldehyde synthesis. Results showed that when SsLPPS underwent a single mutation at positions 730L, D374L, N674L, or G379M, the perillaldehyde yield increased by 16.1%, 9.16%, 13.0%, and 19.46%, respectively, compared to before the mutation. Further combined mutations of two, three, or four positions gradually increased perillaldehyde yield. The most significant yield increase, 2.58 times that of the control group, occurred when all three mutations were present.

[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 26.11 g / L. This yield is higher than the highest level recorded in current technology. Detailed Implementation

[0042] 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.

[0043] In this invention, for the purpose of using fragrant perilla ( Salvia sclarea Wild-type lysine pyrophosphate diol ester synthase from [source name missing] was mutated and optimized to obtain SsLPPs mutants containing at least one mutation among D730L, D374L, N674L, or G379M. Simultaneously, the basal bacteria synthesizing basal alcohol were modified by enhancing the acetyl-CoA synthesis pathway, the MVA pathway, and the perillaldehyde synthesis pathway, and by knocking out some transcriptional repressors. Finally, the SsLPPs mutants were introduced into these basal bacteria, resulting in a significant increase in basal alcohol production.

[0044] 1. Nomenclature of amino acids and DNA nucleic acid sequences

[0045] (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.

[0046] (2) Identification (naming) principles of SsLPPs mutants

[0047] The mutated amino acid in the SsLPP mutant is represented by "original amino acid residue + residue position + substituted amino acid residue". For example, D730L indicates that the amino acid at position 730 is replaced by leucine (L) instead of aspartic acid (D) in wild-type SsLPPs. The position number corresponds to the amino acid sequence number of wild-type SsLPPs in SEQ ID NO.1. The information is shown in Table 1 below:

[0048] Table 1

[0049]

[0050] This invention screens for amino acid mutations in SsLPPs and found that mutations at the D730L, D374L, N674L, and G379M positions are beneficial for increasing the yield of perillyl alcohol.

[0051] Therefore, proteins that, while retaining the D730L, D374L, N674L, or G379M amino acid mutations, exhibit a certain degree of homology with the mutants in Table 1 by mutating the remaining 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.

[0052] Homology refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences.

[0053] 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.

[0054] In this invention, the wild-type SsLPPs are derived from Perilla frutescens ( Salvia sclarea The amino acid sequence is shown in SEQ ID NO.1:

[0055] MTSVNLSRAPAAITRRRLQLQPEFHAECSWLKSSSKHAPLTLSCQIRPKQLSQIAELRVTSLDASQASEKDISLVQTPHKVEVNEKIEESIEYVQNLMTSGDGRISVSPYDTAVIALIKDLKGRDAPQFPSCLEWIAHHQLADGSWGDEFFCIYDRILNTLACVVALKSWNLHSDIIEKGVTYIKENVHKLKGAN VEHRTAGFELVVPTFMQMATDLGIQDLPYDHPLIKEIADTKQQRLKEIPKDLVYQMPTNLLYSLEGLGDLEWERLLKLQSGNGSFLTSPSSTAAVLMHTKDEKCLKYIENALKNCDGGAPHTYPVDIFSRLWAIDRLQRLGISRFFQHEIKYFLDHIESVWEETGVFSGRYTKFSDIDDTSMGVRLLKMHGYDVDP NVLKHFKQQDGKFSCYIGQSVESAPMYNLYRAAQLRFPGEEVLEEATKFAFNFLQEMLVKDRLQERWVISDHLFDEIKLGLKMPWYATLPRVEAAYYLDHYAGSGDVWIGKSFYRMPEISNDTYKELAILDFNRCQTQHQLEWIHMQEWYDRCSLSEFGISKRELLRSYFLAAATIFEPERTQERLLWAKTRILS KMITSFVNISGTTLSLDYNFNGLDEIISSANEDQGLAGTLLATFHQLLDGFDIYTLHQLKHVWSQWFMKVQQGEGSGGEDAVLLANTLNICAGLNEDVLSNNEYTALSTLTNKICNRLAQIQDNKILQVVDGSIKDKELEQDMQALVKLVLQENGGAVDRNIRHTFLSVSKTFYYDAYHDDETTDLHIFKVLFRPVV

[0056] 2. All raw materials and reagents involved in the experiments of this invention can be purchased commercially.

[0057] Chassis cells: Saccharomyces cerevisiae ( S. cerevisiae CEN.PK2-1C. This application uses *Saccharomyces cerevisiae* (CEN.PK2-1C). S. cerevisiae The strain CEN.PK2-1C (from the China Microbial Culture Bank, number Bio-116324) was used as the starting strain for construction and modification.

[0058] 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.

[0059] Plasmid pIYC04 was purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0060] Plasmid pSPGM1 was purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0061] 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:

[0062] 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).

[0063] 3. Information on some of the culture media used in this application:

[0064] The SC-URA plate culture medium consists of: 20 g / L glucose, 15 g / L agar powder, 6.7 g / L yeast amino acid-free nitrogen source (YNB), 0.02 g / L histidine, 0.1 g / L leucine, and 0.02 g / L tryptophan.

[0065] The composition of SC plate culture medium containing 5-FOA (5-fluoroorotic acid) is as follows: 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, and agar powder 15 g / L.

[0066] YPD plate culture medium composition: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agar powder 15 g / L.

[0067] 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.

[0068] 4. The sequence information of some genes or enzymes involved in this application is shown in Table 2.

[0069] Table 2

[0070]

[0071] 5. Some primer information involved in the embodiments of this application is shown in Table 3.

[0072] Table 3

[0073]

[0074] The present invention will be further explained and illustrated below through specific embodiments.

[0075] Example 1: Chassis Microbial Optimization

[0076] I. Overexpression of ADH2, ALD6, and ACS (enhancing the acetyl-CoA synthesis pathway)

[0077] With brewer's yeast ( S. cerevisiae Using CEN.PK2-1C as the starting chassis cells, overexpression of ADH2, ALD6, and ACS yielded strain PKC001. The specific construction steps are as follows:

[0078] 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.

[0079] 1. Design plasmid pCas9-1622b-308a

[0080] Using CRISPOR (http: / / crispor.tefor.net / ) to predict and design brewing yeast ( S. cerevisiae Guide RNA sequences at sites 1622b and 308a on the CEN.PK2-1C genome were obtained, and gRNA sequences with high efficiency and no 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. After PCR amplification using plasmid pScURA as a template, a gene fragment containing gRNAs at sites 1622b and 308a and the selection marker Ura was obtained. The gene fragment obtained by the above PCR amplification was then ligated to the pCas9 plasmid using homologous recombinase to obtain the plasmid pCas9-1622b-308a.

[0081] 2. Design Donor DNA

[0082] (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;

[0083] (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.

[0084] 3. The Donor DNA fragments 1 and 2 obtained in steps 1 and 2 were co-transformed with pCas9-1622b-308a plasmid by electroporation. S. cerevisiae CEN.PK2-1C competent cells were used to integrate the target gene expression cassette using homologous recombination within *Saccharomyces cerevisiae*. The cells were then 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 colonies growing on SC plates containing 5-FOA were simultaneously streaked onto YPD and SC-URA plates and incubated at 30°C. Bacteria that grew on YPD plates but not on SC-URA plates were identified as gene-edited bacteria. These single colonies were selected and incubated on YPD liquid medium at 30°C for 24 h. The culture was then preserved (with 20% glycerol) to obtain the chassis strain PKC001, which was stored at -80°C (with 20% glycerol) for future use. The validation primers were V-1622b-F / R and V-308a-F / R.

[0085] II. Overexpression of tHMG1, IDI, and Erg20 (enhancing the MVA pathway)

[0086] 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).

[0087] 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.

[0088] The Erg20 encoding gene was inserted at site 911b of the genome, and the donor DNA was up-TPGK1-PGAL2-PGAL7-ERG20-TTPS1-down.

[0089] 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.

[0090] III. Knockout of GAL80, ROX1, and DOS2 (knockout of negative transcriptional regulators)

[0091] 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.

[0092] 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).

[0093] 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).

[0094] 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).

[0095] IV. Introducing gerany-gerany-pyrophosphate synthase GGPPS (CrtE) from Pharfia redis to enhance GGPP synthesis flux.

[0096] 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.

[0097] 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.

[0098] Example 2: Construction of SsLPPS mutant plasmid

[0099] The SsLPPS 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.

[0100] I. Construction of pIYC04-SsLPPS plasmid

[0101] Using pIYC04 as the expression vector, recombinant expression plasmids were constructed using the In-Fusion strategy.

[0102] Using the DNA sequence synthesized according to SEQ ID NO.6 as a template, and LPPS-F and LPPS-R as primers, PCR amplification was performed. SsLPPS Gene fragments; using pIYC04 vector as a template and TADH-F and PTEF-R as primers, the linearized pIYC04 vector was amplified by PCR. Following the kit instructions, the obtained gene fragments were... SsLPPS The gene fragment and the linearized pIYC04 vector fragment underwent homologous recombination reaction. After reacting at 50℃ for 1 hour, the mixture was transformed. E.coli DH5α competent cells were plated on Amp-resistant plates and cultured overnight at 37°C. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB liquid medium containing Amp. Sequencing was performed to verify the results, and the recombinant plasmid pIYC04-SsLPPS with correct sequencing results was extracted for later use.

[0103] two, SsLPPS Construction of mutant plasmids

[0104] Homology modeling of SsLPPS was performed using AlphaFold3, and then the substrate GGPP was docked to the active site of SsLPPS protein using the molecular docking software AutoDock. By analyzing the amino acid residues and steric hindrance of the receptor-ligand interaction, D730L, D374L, N674L, and G379M were identified as key amino acid residues to be mutated.

[0105] Using pIYC04-SsLPPS as a template, mutant primers (LPPS-D730L-F and LPPS-D730L-R) were designed based on the D730L single-point mutation for PCR amplification. The PCR product was digested with the restriction endonuclease DpnI for 2 hours to remove the template. After agarose gel electrophoresis, the target band was recovered by elution with ddH2O and then transformed. E.coli DH5α competent cells were plated on Amp-resistant plates and cultured overnight at 37°C. 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 correct plasmid pIYC04-SsLPPS(D730L) was extracted for later use.

[0106] The construction methods for the remaining plasmids are the same as for the D730L 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:

[0107] pIYC04-SsLPPS (D374L);

[0108] pIYC04-SsLPPS (N674L);

[0109] pIYC04-SsLPPS (G379M);

[0110] pIYC04-SsLPPS (D730L, D374L);

[0111] pIYC04-SsLPPS (D730L, N674L);

[0112] pIYC04-SsLPPS (D730L, G379M);

[0113] pIYC04-SsLPPS (D730L, D374L, N674L);

[0114] pIYC04-SsLPPS (D730L, D374L, G379M);

[0115] pIYC04-SsLPPS (D730L, D374L, N674L, G379M).

[0116] Example 3 SsSCS expression plasmid construction

[0117] To construct a complete synthetic pathway for perillyl alcohol, further construction is needed. SsSCS Expression plasmid.

[0118] The SsSCS encoding gene was codon-optimized for Saccharomyces cerevisiae (the optimized nucleic acid sequence is shown in SEQ ID NO. 7), and the full-length DNA sequence was synthesized.

[0119] Using pSPGM1 as the expression vector, recombinant expression plasmids were constructed using the In-Fusion strategy.

[0120] Using the DNA sequence synthesized according to SEQ ID NO.7 as a template, and SCS-F and SCS-R as primers, PCR amplification was performed. SsSCS Gene fragments; using 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 gene fragments were... SsSCS The gene fragment and the linearized pSPGM1 vector fragment underwent homologous recombination, and after incubation at 50°C for 1 hour, the mixture was transformed. E.coli DH5α competent cells were plated on Amp-resistant plates and cultured overnight at 37°C. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB liquid medium containing Amp. Sequencing was performed to verify the results, and the recombinant plasmid pSPGM1-SsSCS with correct sequencing results was extracted for later use.

[0121] Example 4: Construction of recombinant strains (SsLPPS / mutant, SsSCS plasmid expression)

[0122] 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 or their mutants, together with SsSCS, constitute a complete perillaldehyde synthesis pathway) to obtain recombinant strains CSL001-CSL010 and control strain CON-CS01, which were frozen at -80°C for later use. Specific information is shown in Table 4 below.

[0123] Table 4

[0124]

[0125] Example 5: Perillaldehyde Production Experiment (SsLPPS / Mutant, SsSCS Plasmid Expression)

[0126] Shake-flask fermentation:

[0127] Frozen strains CSL001-CSL010 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.

[0128] Table 5

[0129]

[0130] 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 CSL001-CSL010 expressing SsLPPS mutant and wild-type SsSCS after 120 h of fermentation reached 474.14 mg / L-1124.7 mg / L, representing an increase of 9.16%-158.00% compared to the control group. Specifically, when SsLPPS underwent single mutations at positions 730L, D374L, N674L, and G379M, the yield increased by 16.1%, 9.16%, 13.0%, and 19.46%, respectively. Based on this, further combined mutations of two, three, and four sites can gradually increase the yield of perillaldehyde. When mutations occur at the 730L, D374L, N674L, and G379M sites, the yield increase is the most significant, which is 2.58 times that of the control group.

[0131] Example 6: Construction of Recombinant Strains (SsLPPS / Mutant, SsSCS Genome Integration)

[0132] To further improve the genetic stability of the recombinant strain, SsLPPS or its mutants, as well as SsSCS, were integrated into the genome.

[0133] 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).

[0134] The insertion site for the coding gene of SsLPPS or its mutant, and SsSCS, is the 208a site of the genome, and the donor DNA is up-TCYC1-SsLPPS / mutant-PGAL1-PGAL10-SsSCS-TADH1-down.

[0135] 2. The knockout plasmid pCas9-208a and donor DNA: up-TCYC1-SsLPPS / mutant-PGAL1-PGAL10-SsSCS-TADH1-down were co-transformed into strain PKC005 to obtain perillaldehyde synthesizing strains CSL011-CSL020 (see Table 6 for details) and control strain CON-CS02. The strains were stored for later use (20% glycerol).

[0136] Table 6

[0137]

[0138] Example 7: Perillyl alcohol production experiment (SsLPPS / mutation, SsSCS genome integration)

[0139] 1. Shake-flask fermentation:

[0140] The frozen strains CSL011-CSL020 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.

[0141] Table 7

[0142]

[0143] As can be seen from the data in Table 7, the yield of genome-integrated SsLPPS / mutant and SsSCS genes is significantly higher than that of plasmid expression in Table 5, but the overall trend remains the same.

[0144] Furthermore, the perillaldehyde yield of the recombinant control strain CON-CS02 expressing wild-type SsLPPS and SsSCS reached 567.48 mg / L after 120 h of fermentation, while the perillaldehyde yield of the recombinant strains CSL011-CSL020 expressing SsLPPS mutant and wild-type SsSCS reached 607.30 mg / L-1535.89 mg / L after 120 h of fermentation, representing an increase of 7.02%-170.65% compared to the control group. Specifically, when SsLPPS underwent single mutations at positions 730L, D374L, N674L, and G379M, the yield increased by 9.32%, 7.02%, 8.18%, and 14.77%, respectively. Further combined mutations of two, three, and four sites can gradually increase the yield of sagerol. The most significant increase was observed when mutations occurred at the 730L, D374L, N674L, and G379M sites, which was 2.7 times that of the control group.

[0145] 2.5L fermentation tank:

[0146] The SsLPPS tetrastrain CSL020, 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 DO levels adjusted in tandem. Aeration was maintained at 1 vvm and pressure at 0.02 MPa.

[0147] 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.

[0148] After 24 hours of fermentation, 10% (v / v) dodecane solvent was added for biphase fermentation.

[0149] The composition of the fermentation medium, fed medium, and trace elements is shown in Table 8 below.

[0150] Table 8

[0151]

[0152] The fermentation cycle was 120 h. Samples were taken to test the yield of perillaldehyde. The yields of the three parallel fermentation experiments were 25.56, 26.31 and 26.47 g / L, respectively, with an average yield of 26.11 g / L.

[0153] 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 mutant of lysine pyrophosphate diol ester synthase, characterized in that, The mutant is any one of the following (1)-(7): (1) The mutant is a mutant obtained by D730L mutation on the basis of wild-type lysine pyrophosphate diol ester synthase shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; (2) The mutant is a mutant obtained by D730L and D374L mutations on the basis of wild-type lysine pyrophosphate diol ester synthase shown in SEQ ID NO.1; (3) The mutant is a mutant obtained by D730L and N674L mutations on the basis of wild-type lysine pyrophosphate diol ester synthase shown in SEQ ID NO.1; (4) The mutant is a mutant obtained by D730L and G379M mutations on the basis of wild-type lysine pyrophosphate diol ester synthase shown in SEQ ID NO.1; (5) The mutant is a mutant obtained by D730L, D374L and N674L mutations on the basis of wild-type lysine pyrophosphate synthase shown in SEQ ID NO.1; (6) The mutant is a mutant obtained by D730L, D374L and G379M mutations on the basis of wild-type lysine pyrophosphate diol ester synthase shown in SEQ ID NO.1; (7) The mutant is a mutant obtained by D730L, D374L, N674L and G379M mutations on the basis of wild-type lysine pyrophosphate synthase shown in SEQ ID NO.

1.

2. The application of the lysine pyrophosphate synthase mutant of claim 1 in the production of perillyl alcohol, or in the catalytic generation of lysine pyrophosphate from gerany-gerany-pyrophosphate.

3. The encoding gene of the lysine pyrophosphate diol ester 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 lysine pyrophosphate diol ester 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 perilla syringol synthase SsSCS; (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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