Recombinant saccharomyces cerevisiae for producing decaprenylphenol and construction method and application thereof

Abstract

The application discloses a recombinant saccharomyces cerevisiae for producing decaprenylphenol and a construction method and application thereof, and belongs to the technical field of microorganisms. Rhodobacter sphaeroides The recombinant saccharomyces cerevisiae is obtained by expressing a decaprenyl pyrophosphate synthase gene from Rhodobacter sphaeroides in the saccharomyces cerevisiae, overexpressing a phospholipase gene of endogenous phosphatidic acid of the saccharomyces cerevisiae, and down-regulating a transcription level of a squalene synthesis gene ERG9 and strengthening an endogenous MVA pathway. 10 The application realizes the production of decaprenylphenol in the saccharomyces cerevisiae for the first time, and the yield reaches 112 mg / L in a flask; decaprenylphenol is extracted from the recombinant saccharomyces cerevisiae through fermentation, and coenzyme Q 10 is synthesized through a chemical method, compared with traditional fermentation of Rhodobacter sphaeroides for producing coenzyme Q 10 , the method has the advantages of high fermentation stability, simple culture medium and the like, and has industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a recombinant Saccharomyces cerevisiae that produces decanopentenol through genetic engineering, its construction method, and its application. Background Technology

[0002] Coenzyme Q 10 (CoQ) 10 α-hydroxyquinone (HHQ) is a lipid-soluble quinone compound widely found in the inner mitochondrial membrane and is a key component of cellular energy metabolism (ATP synthesis) and the antioxidant defense system. Since its discovery in 1957, its applications have expanded from medicine (such as adjunctive treatment for cardiovascular diseases and neurodegenerative diseases) to health products, functional foods, and cosmetics.

[0003] Currently, Coenzyme Q 10 The main extraction methods for coenzyme Q include animal cell extraction, semi-synthesis of solanesol from tobacco leaves, complete synthesis, microbial fermentation, and plant cell culture. Among these, microbial fermentation has become a global research hotspot in recent years and is considered the most promising production method. Conventional microbial fermentation methods are used to produce coenzyme Q. 10 The method involves fermentation production using Rhodopseudomonas aeruginosa. Through mutagenesis, genetic engineering, and optimization of fermentation conditions, the current large-scale fermentation concentration of Rhodopseudomonas aeruginosa in industrial applications is around 3 g / L.

[0004] Decaprenol is an open-chain terpene compound with the basic structural unit of isopentenene, and its structure is as follows: Decopentenol is synthesized using a direct side-chain introduction method for coenzyme Q. 10 An important intermediate, this invention proposes: preparing decanoisopentenol via microbial fermentation, and then obtaining coenzyme Q through chemical synthesis. 10 It is another efficient way to produce coenzyme Q. 10 The method.

[0005] Saccharomyces cerevisiae has a high MVA (mevaleric acid) flux, making it highly suitable for producing terpenoids. Several successful terpenoid products have already entered the large-scale production stage. However, yeast is a eukaryote, and modifying yeast to directly produce coenzyme Q presents a challenge. 10 The synthesis pathway is complex, and there are still problems such as blockages in multiple organelles, such as mitochondria, which are not yet fully understood. Therefore, modifying yeast to produce coenzyme Q presents challenges. 10 The precursor decanopentenol can avoid the problem of long and difficult-to-enhance pathways after coenzyme Q10 in yeast, making it a more promising solution. Therefore, it is necessary to study how to construct a recombinant Saccharomyces cerevisiae strain capable of efficiently fermenting and producing decanopentenol. To date, no literature reports the production of decanopentenol using Saccharomyces cerevisiae. In view of this, the present invention provides the following technical solution. Summary of the Invention

[0006] Microorganisms possess advantages such as rapid growth, short fermentation cycles, and low fermentation costs, and their clear genetic background facilitates genetic modification. This invention aims to achieve rapid and large-scale production of decanopenol by recombining the biosynthetic pathway of decanopenol in microbial chassis cells, providing a new method for the development and application of decanopenol. Specifically, this invention aims to modify a recombinant *Saccharomyces cerevisiae* strain capable of efficiently producing decanopenol using genetic engineering technology. By fermenting this recombinant strain, low-cost, large-scale industrial production of decanopenol can be achieved, and coenzyme Q can be further synthesized chemically. 10 Another object of the present invention is to provide a method for constructing the recombinant Saccharomyces cerevisiae and its application in the fermentation production of decanpentol.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant Saccharomyces cerevisiae strain, characterized in that, compared with the original Saccharomyces cerevisiae strain, the recombinant Saccharomyces cerevisiae strain has the following characteristics:

[0009] I) Expression of Rhodotorula eugenolide-derived decanopene pyrophosphate synthase RsDDSA; and / or

[0010] II) Expressing RsDDSA, a lipid droplet-localizing cyclophosphamide-derived decanopentene pyrophosphate synthase.

[0011] Unexpectedly, this invention has discovered that localizing the decanopene pyrophosphate synthase RsDDSA derived from Rhodophytae bacteria onto lipid droplets significantly improves the production rate of decanopene alcohol by recombinant Saccharomyces cerevisiae. Although the specific embodiments of this invention only achieved lipid droplet localization of the decanopene pyrophosphate synthase RsDDSA derived from Rhodophytae bacteria, those skilled in the art should understand that decanopene pyrophosphate synthases derived from other strains (such as Agrobacterium tumefaciens) that can be successfully expressed in Saccharomyces cerevisiae can also be localized onto lipid droplets and achieve the technical effects described in the embodiments of this invention.

[0012] In some embodiments of the present invention, a fusion gene of the lipid droplet localization gene and RsDDSA is constructed by fusing the lipid droplet localization gene with the decanopyrene pyrophosphate synthase RsDDSA gene derived from Rhodotorula globulus, and the fusion gene is then integrated into Saccharomyces cerevisiae to achieve lipid droplet localization.

[0013] In this invention, the lipid droplet localization gene is selected from one of the N-terminal signal peptide of the PLN1 gene, AAMB, and Olesin. Preferably, the lipid droplet localization gene is selected from the N-terminal signal peptide of the PLN1 gene, and its nucleotide sequence is shown in SEQ ID NO:41.

[0014] Furthermore, compared to the original Saccharomyces cerevisiae strain, the recombinant Saccharomyces cerevisiae strain also overexpresses the endogenous dephosphatase LPP1 of Saccharomyces cerevisiae.

[0015] Furthermore, compared to the original yeast strain, the recombinant Saccharomyces cerevisiae is characterized by downregulation of the transcriptional level of squalene synthase ERG9.

[0016] Furthermore, the recombinant Saccharomyces cerevisiae, compared to the original yeast strain, also includes an enhanced MVA pathway, specifically, overexpression of MVA pathway genes. In a preferred embodiment of the present invention, the genes for the MVA pathway are selected from ERG10, ERG13, tHMGR, ERG12, and ERG20.

[0017] In a specific embodiment of the present invention, the recombinant Saccharomyces cerevisiae has a combination of the features shown in 1)-5):

[0018] 1) Expressing the RsDDSA gene, a decanpentene pyrophosphate synthase derived from Rhodococcus globulus;

[0019] 2) Overexpression of the endogenous dephosphatase LPP1 gene;

[0020] 3) Inhibit the squalene synthase ERG9 gene;

[0021] 4) Overexpression of MVA pathway genes ERG10, ERG13, tHMGR, ERG12, and ERG20;

[0022] 5) Expressing the RsDDSA gene of decanopyrene pyrophosphate synthase derived from Rhodotorula globulus, which localizes lipid droplets.

[0023] In this invention, the RsDDSA gene is optimized according to the codon preference of Saccharomyces cerevisiae. In some embodiments of this invention, the RsDDSA gene contains a nucleotide sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1.

[0024] In one specific embodiment of the present invention, the RsDDSA gene has the nucleotide sequence shown in SEQ ID NO:1.

[0025] The NCBI number for the endogenous dephosphorylase LPP1 gene of the Saccharomyces cerevisiae is NM_001180811.3.

[0026] This invention enables the overexpression of the decanopyrene pyrophosphate synthase RsDDSA gene from Rhodotorula globulus, the endogenous dephosphorylase LPP1 gene from Saccharomyces cerevisiae, the MVA pathway gene, and the lipid droplet-localized decanopyrene pyrophosphate synthase RsDDSA gene from Rhodotorula globulus.

[0027] In some embodiments of the present invention, the present invention achieves overexpression of the target gene in recombinant bacteria through gene integration. Methods for integrating the target gene fragment into the host cell genome are well known to those skilled in the art; for example, homologous recombination can be used to integrate the target gene fragment into a target site in the host cell genome. Homologous recombination relies on the homology between DNA molecules. When applying homologous recombination, it is usually necessary to add upstream and downstream sequences homologous to the target site, upstream and downstream of the exogenous DNA sequence or the target gene fragment. These upstream and downstream sequences are the upstream homologous arm and downstream homologous arm described in specific embodiments of the present invention.

[0028] In some embodiments of the present invention, the RsDDSA gene is integrated into the GAL1-7 gene site and rDNA site in the Saccharomyces cerevisiae genome via homologous recombination, and the RsDDSA gene is expressed by the promoter pGAL1. In a specific embodiment of the present invention, the target gene used to integrate the RsDDSA gene into the above-mentioned target site is selected from the RsDDSA gene expression cassette.

[0029] In some embodiments of the present invention, the LPP1 gene is integrated into an rDNA site in the Saccharomyces cerevisiae genome via homologous recombination to express the endogenous dephosphorylase LPP1 gene of Saccharomyces cerevisiae via the promoter pGAL10. In a specific embodiment of the present invention, the target gene used to integrate the LPP1 gene into the target site is selected from the LPP1 gene expression cassette.

[0030] In some embodiments of the present invention, the endogenous genes ERG10 and tHMGR of the MVA pathway are integrated into the 308a site of the Saccharomyces cerevisiae genome via homologous recombination; the ERG12 and ERG13 genes are integrated into the 1114a site of the Saccharomyces cerevisiae genome via homologous recombination; and the ERG20 and tHMGR genes are integrated into the 1021b site of the Saccharomyces cerevisiae genome via homologous recombination.

[0031] In some embodiments of the present invention, the lipid droplet-localized RsDDSA gene, namely the PLN1-RsDDSA fusion gene, is integrated into the 911b site of the Saccharomyces cerevisiae genome through homologous recombination mechanism, and the lipid droplet-localized RsDDSA gene is expressed by the promoter pGAL1.

[0032] In one specific embodiment of the present invention, the characteristics of the recombinant Saccharomyces cerevisiae are achieved in the following manner:

[0033] 1) Integration of RsDDSA gene expression cassette

[0034] In this invention, the RsDDSA gene is integrated into the GAL1-7 gene locus in the Saccharomyces cerevisiae genome, and the genes corresponding to this locus are GAL1, GAL10, and GAL7.

[0035] In some implementations, the RsDDSA gene is integrated into the GAL1-7 gene in the Saccharomyces cerevisiae genome, or upstream or downstream thereof.

[0036] In some preferred embodiments, the RsDDSA gene is integrated into the GAL1-7 gene in the Saccharomyces cerevisiae genome, or within 1000 bp upstream or downstream of it.

[0037] In some preferred embodiments, the RsDDSA gene is integrated into the range of 1000 bp upstream to 1000 bp downstream of the GAL1-7 gene in the Saccharomyces cerevisiae genome.

[0038] In this invention, the target gene for integrating the RsDDSA gene into the target site includes an RsDDSA gene expression cassette. In one specific embodiment of this invention, the RsDDSA gene expression cassette is pGAL1-RsDDSA-tCYC1.

[0039] In some implementations, the N-terminus and C-terminus of the RsDDSA gene expression cassette contain homologous arms, respectively.

[0040] In some embodiments, the homologous arm comprises at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000 bp sequence range upstream of the GAL1-7 gene in the Saccharomyces cerevisiae genome.

[0041] In some embodiments, the homologous arm comprises at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000 bp sequence range downstream of the GAL1-7 gene in the Saccharomyces cerevisiae gene.

[0042] In some preferred embodiments, the expression cassette of the RsDDSA gene includes the N-terminal homologous arm (upstream homologous arm) as a basis. Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the homologous arm GAL1-7-UP was amplified by PCR using primers GAL1-7-UF / GAL1-7-UR (sequences shown in SEQ ID NO:5 and SEQ ID NO:6).

[0043] In some preferred embodiments, the expression cassette C-terminal homologous arm (downstream homologous arm) of the RsDDSA gene is included as a basis for... Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the homologous arm GAL1-7-Dn was amplified by PCR using primers GAL1-7-DF / GAL1-7-DR (sequences shown in SEQ ID NO:7 and SEQ ID NO:8).

[0044] 2) Integration of RsDDSA and LPP1 gene expression cassettes

[0045] In this invention, the target gene for integrating the RsDDSA and LPP1 genes into the target site includes an RsDDSA and LPP1 gene expression cassette. In one specific embodiment of this invention, the RsDDSA and LPP1 gene expression cassette is tCYC1-RsDDSA-pGAL1-pGAL10-LPP1-tADH1.

[0046] The RsDDSA and LPP1 gene expression cassettes are integrated into the rDNA sites in the Saccharomyces cerevisiae genome.

[0047] In some implementations, the RsDDSA and LPP1 gene expression cassettes are integrated into rDNA sites in the Saccharomyces cerevisiae genome, either upstream or downstream of them.

[0048] In some preferred embodiments, the RsDDSA and LPP1 gene expression cassettes are integrated into the rDNA site in the Saccharomyces cerevisiae genome, or within 1000 bp upstream or downstream of it.

[0049] In some preferred embodiments, the RsDDSA and LPP1 gene expression cassettes are integrated into the rDNA site in the Saccharomyces cerevisiae genome within a range of 1000 bp upstream to 1000 bp downstream.

[0050] In some implementations, the N-terminus and C-terminus of the RsDDSA and LPP1 gene expression cassettes contain homologous arms, respectively.

[0051] In some embodiments, the homologous arm comprises at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000 bp sequence range upstream of an rDNA site in the Saccharomyces cerevisiae genome.

[0052] In some embodiments, the homologous arm comprises at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000 bp sequence range downstream of an rDNA site in the Saccharomyces cerevisiae genome.

[0053] In some preferred embodiments, the N-terminal homologous arms of the RsDDSA and LPP1 gene expression cassettes are... Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the homologous arm rDNA-UP was amplified by PCR using primers rDNA-UF / rDNA-UR (sequences shown in SEQ ID NO:23 and SEQ ID NO:24).

[0054] In some preferred embodiments, the C-terminal homologous arms of the RsDDSA and LPP1 gene expression cassettes are... Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the homologous arm rDNA-Dn was amplified by PCR using primers rDNA-DF / rDNA-DR (sequences shown in SEQ ID NO:25 and SEQ ID NO:26).

[0055] 3) Inhibit the expression of squalene synthase ERG9

[0056] To achieve downregulation of the transcriptional level of squalene synthase ERG9, the present invention preferably initiates the expression of the squalene synthase ERG9 gene with a weak promoter, wherein the weak promoter has a relative strength of up to 20%, up to 10%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1% relative to the original promoter pERG9 promoter in recombinant Saccharomyces cerevisiae.

[0057] In one specific embodiment of the present invention, the squalene synthase ERG9 gene is expressed by a weak promoter pHXT1, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0058] 4) Overexpression of MVA pathway genes ERG10, ERG13, tHMGR, ERG12, and ERG20

[0059] The NCBI numbers of the genes in the MVA pathway mentioned above are: ERG10 (Gene ID: 856079), ERG13 (Gene ID: 854913), tHMGR (Gene ID: 854900), ERG12 (Gene ID: 855248), and ERG20 (Gene ID: 853272).

[0060] Those skilled in the art can achieve overexpression of endogenous genes via the MVA pathway using conventional techniques. In a specific embodiment of the present invention, the ERG10 and tHMGR genes are integrated into the 308a site of the *Saccharomyces cerevisiae* genome using the ERG10 and tHMGR gene expression cassette; the ERG12 and ERG13 genes are integrated into the 1114a site of the *Saccharomyces cerevisiae* genome using the ERG12 and ERG13 gene expression cassette; and the ERG20 and tHMGR genes are integrated into the 1021b site of the *Saccharomyces cerevisiae* genome using the ERG20 and tHMGR gene expression cassette.

[0061] Furthermore, the N-terminus and C-terminus of the aforementioned gene expression cassette each contain homologous arms, which can be obtained by those skilled in the art through specific gene loci using conventional technical means.

[0062] 5) Expression of the RsDDSA gene for lipid droplet localization

[0063] In some embodiments of the present invention, the N-terminal signal peptide gene of the PLN1 gene is selected as the lipid droplet localization gene. Specifically, the nucleotide sequence of the N-terminal signal peptide gene of the PLN1 gene is shown in SEQ ID NO:41. The N-terminal signal peptide gene of the PLN1 gene is fused with the decanpentene pyrophosphate synthase RsDDSA gene derived from Rhodotorula glutinis to construct a fusion gene.

[0064] In some embodiments, the fusion gene, PLN1-RsDDSA, is integrated into the 911b site gene in the Saccharomyces cerevisiae genome, or upstream or downstream thereof.

[0065] In some preferred embodiments, the fusion gene is integrated into the 911b site gene in the Saccharomyces cerevisiae genome within a range of 1000 bp upstream to 1000 bp downstream.

[0066] In this invention, the target gene used to integrate the fusion gene into the target site is the PLN1-RsDDSA gene expression cassette. In one specific embodiment of this invention, the PLN1-RsDDSA gene expression cassette is pGAL1-PLN1-RsDDSA-tCYC1.

[0067] In some embodiments, the N-terminus and C-terminus of the PLN1-RsDDSA gene expression cassette contain homologous arms, respectively.

[0068] In some embodiments, the homologous arm comprises at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000 bp sequence range upstream of the 911b site gene in the Saccharomyces cerevisiae genome.

[0069] In some embodiments, the homologous arm comprises at least 800, at least 850, at least 900, at least 950, or at least 1000 consecutive nucleotides within a 1000 bp sequence range downstream of the 911b site gene in the Saccharomyces cerevisiae gene.

[0070] In some preferred embodiments, the expression cassette of the PLN1-RsDDSA fusion gene includes the N-terminal homologous arm (upstream homologous arm) as a basis. Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the homologous arm 911b-UP was amplified by PCR using primers 911b-UF / 911b-UR (sequences shown in SEQ ID NO:49 and SEQ ID NO:50).

[0071] In some preferred embodiments, the expression cassette C-terminal homologous arm (downstream homologous arm) of the PLN1-RsDDSA fusion gene is included as a basis for... Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the homologous arm 911b-Dn was amplified by PCR using primers 911b-DF / 911b-DR (sequences shown in SEQ ID NO:51 and SEQ ID NO:52).

[0072] In some embodiments of the present invention, in order to enhance the expression of the target protein, those skilled in the art can increase the copy number of multiple target genes integrated into the Saccharomyces cerevisiae genome.

[0073] Unless otherwise specified, the starting strain of brewing yeast described in this invention is brewing yeast CEN.PK2-1D.

[0074] In one specific embodiment of the present invention, the recombinant Saccharomyces cerevisiae provided by the present invention is Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae DOH15 was deposited at the China Center for Type Culture Collection (CCTCC) on May 22, 2025, with accession number CCTCC NO: M 20251155.

[0075] Secondly, a method for constructing a recombinant Saccharomyces cerevisiae strain that produces decanopentenol, characterized by comprising the following modifications to the starting Saccharomyces cerevisiae strain:

[0076] S1) expresses the RsDDSA gene, a decanpentene pyrophosphate synthase derived from Rhodophyta globulinii; and

[0077] S2) Overexpression of the endogenous dephosphatase LPP1 gene in Saccharomyces cerevisiae; and

[0078] S3) Downregulated the transcriptional level of squalene synthase ERG9 in the cell matrix of Saccharomyces cerevisiae; and

[0079] S4) Overexpression of MVA pathway genes ERG10, ERG13, tHMGR, ERG12, and ERG20; and

[0080] S5) expresses the RsDDSA gene, a lipid droplet-localized cyclophosphamide-derived decanopyrene pyrophosphate synthase.

[0081] In some embodiments of the present invention, the method for constructing a recombinant Saccharomyces cerevisiae producing decanopentenol includes the following steps:

[0082] 1) Construct the RsDDSA gene expression cassette and integrate it into the GAL1-7 site of the starting yeast strain through homologous recombination to obtain yeast strain DOH01;

[0083] 2) Construct RsDDSA and LPP1 gene expression cassettes, and integrate the RsDDSA and LPP1 gene expression cassettes into the rDNA site of the chromosome of yeast strain DOH01 through homologous recombination mechanism to obtain yeast strain DOH03;

[0084] 3) Based on yeast strain DOH03, the ERG9 promoter was replaced with the pHXT1 promoter to obtain yeast strain DOH04;

[0085] 4) Construct ERG10 and tHMGR gene expression cassettes, ERG12 and ERG13 gene expression cassettes and ERG20 and tHMGR gene expression cassettes, and integrate the gene expression cassettes into the 308a, 1114a and 1021b sites of the yeast strain DOH04 chromosome through homologous recombination mechanism to obtain yeast strain DOH07;

[0086] 5) The N-terminal signal peptide gene of the PLN1 gene was fused with the RsDDSA gene to obtain the fusion gene PLN1-RsDDSA. The PLN1-RsDDSA gene expression cassette was constructed and integrated into the 911b site of the chromosome of yeast strain DOH07 through homologous recombination to obtain yeast strain DOH15.

[0087] In one specific embodiment of the present invention, step 1) includes the following steps:

[0088] 1.1) Using plasmid pBBS17 as a vector, construct the RsDDSA expression plasmid pBBS17-RsDDSA;

[0089] 1.2) Using pBBS17-RsDDSA as a template, the RsDDSA gene expression cassette (pGAL1-RsDDSA-tCYC1) with pGAL1 as the promoter was obtained by PCR amplification.

[0090] 1.3) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, homologous arms of the GAL1-7 gene locus were obtained by PCR.

[0091] 1.4) The RsDDSA gene expression cassette and the upstream and downstream homologous arms were integrated into the GAL1-7 site of the starting yeast strain to obtain yeast strain DOH01.

[0092] In one specific embodiment of the present invention, step 2) includes the following steps:

[0093] 2.1) Using plasmid pBBS17-RsDDSA as a vector, the LPP1 gene fragment was inserted to obtain plasmid pBBS17-RsDDSA-LPP1, which expresses both the RsDDSA and LPP1 genes.

[0094] 2.2) Using pBBS17-RsDDSA-LPP1 as a template, the bidirectional promoter expression cassette of RsDDSA and LPP1 genes (tCYC1-RsDDSA-pGAL1-pGAL10-LPP1-tADH1) was obtained by PCR amplification.

[0095] 2.3) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the upstream and downstream homologous arms of the rDNA gene locus were obtained by PCR.

[0096] 2.4) The RsDDSA and LPP1 gene expression cassettes and the upstream and downstream homologous arms were integrated into the rDNA site of the yeast strain DOH01 chromosome to obtain yeast strain DOH03.

[0097] In one specific embodiment of the present invention, step 3) includes the following steps:

[0098] 3.1) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the weak promoter pHXT1 gene fragment was obtained by PCR.

[0099] 3.2) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the upstream and downstream homologous arms of the ProERG9 gene locus were obtained by PCR.

[0100] 3.3) The promoter of the squalene synthase ERG9 gene was replaced with the weak promoter pHXT1 and integrated into the genome of yeast strain DOH03 to obtain yeast strain DOH04.

[0101] In one specific embodiment of the present invention, step 4) includes the following steps:

[0102] 4.1) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, ERG10, ERG13, tHMGR, ERG12 and ERG20 gene fragments were obtained by PCR.

[0103] 4.2) Using plasmid pBBS17 as a vector, construct MVA endogenous pathway expression plasmids pBBS17-ERG10-tHMGR, pBBS17-ERG12-ERG13 and pBBS17-ERG20-tHMGR;

[0104] 4.3) Using the above expression plasmid as a template, the gene expression cassette was obtained by PCR amplification;

[0105] 4.4) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, homologous arms of gene loci 308a, 1114a, and 1021b were obtained by PCR.

[0106] 4.5) The gene expression cassette and the upstream and downstream homologous arms are integrated into the 308a, 1114a, and 1021b sites of the yeast strain DOH04 chromosome to obtain the yeast strain DOH07.

[0107] In one specific embodiment of the present invention, step 5) includes the following steps:

[0108] 5.1) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the N-terminal localization signal peptide gene fragment of the PLN1 gene was obtained by PCR.

[0109] 5.2) The fusion gene PLN1-RsDDSA was obtained by fusion PCR, and the fusion gene expression plasmid pBBS17-PLN1-RsDDSA was constructed using plasmid pBBS17 as a vector.

[0110] 5.3) Using pBBS17-PLN1-RsDDSA as a template, the PLN1-RsDDSA fusion gene expression cassette (pGAL1-PLN1-RsDDSA-tCYC1) was obtained by PCR amplification.

[0111] 5.4) with Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, homologous arms upstream and downstream of the 911b gene locus were obtained by PCR.

[0112] 5.5) The PLN1-RsDDSA fusion gene expression cassette and the upstream and downstream homologous arms were integrated into the 911b site of the yeast strain DOH07 chromosome to obtain yeast strain DOH15.

[0113] Thirdly, the recombinant Saccharomyces cerevisiae described in the first aspect of the present invention or the recombinant Saccharomyces cerevisiae constructed by the method described in the second aspect of the present invention is used in at least one of the following:

[0114] 1) Application in the fermentation production of decanoisopentenol;

[0115] 2) Application in the genetic breeding of microorganisms that produce decanpentol;

[0116] 3) In the production of Coenzyme Q 10 Applications in [the field].

[0117] Fourthly, the present invention provides a method for producing decanoisopentenol by fermentation, characterized in that it includes the step of culturing the recombinant yeast described in the first aspect of the present invention or the recombinant yeast constructed by the method described in the second aspect of the present invention.

[0118] Instructions for Preservation of Microbial Strains

[0119] Strain classification and naming: Saccharomyces cerevisiae DOH15

[0120] Latin name of the strain: Saccharomyces cerevisiae

[0121] Collection Center Registration Number: CCTCC NO: M 20251155

[0122] Preservation Institution: China Center for Type Culture Collection

[0123] Abbreviation for depository institution: CCTCC

[0124] Address of the depository: Wuhan University, Wuhan, China

[0125] Date of preservation: May 22, 2025. Attached Figure Description

[0126] Figure 1 The biosynthetic pathway of decanopentenol;

[0127] Figure 2 pBBS17 plasmid map;

[0128] Figure 3 pBBS17-RsDDSA plasmid map;

[0129] Figure 4 pBBS17-RsDDSA-LPP1 plasmid map;

[0130] Figure 5 pBBS17-PLN1-RsDDSA plasmid map;

[0131] Figure 6 HPLC analysis of the synthesis of decanpentenol by standard and engineered yeast strains (A, standard; B, engineered yeast strain CENPK2 before modification; C, engineered yeast strain DOH03 after modification; D, engineered yeast strain DOH15 after modification).

[0132] Figure 7 A statistical chart showing the yield of decanterenol synthesized by engineered yeast strains. Detailed Implementation

[0133] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0134] Terminology Explanation

[0135] In this invention, the term "starting strain" refers to a strain that is modified through genetic engineering or other means, and can also be called a parent strain. Strains obtained by modifying the starting strain through rational design or by mutagenesis are called genetically engineered bacteria, engineered bacteria, or recombinant bacteria.

[0136] In this invention, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms and can be replaced with “including but not limited to.”

[0137] In this invention, "expression" refers to the process by which a nucleic acid sequence is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into peptides, polypeptides, amino acid sequences, or proteins. If the nucleic acid sequence originates from genomic DNA, expression may include splicing mRNA in eukaryotic cells.

[0138] In this invention, when the term "encoding" is applied to a nucleic acid sequence, it refers to a nucleic acid sequence that, if in its natural state or when manipulated by methods well known to those skilled in the art, can be transcribed to produce mRNA and / or translated to produce a polypeptide, is called a "coding" polypeptide sequence. The antisense strand is the complement of such nucleic acid, from which the coding sequence can be deduced.

[0139] In this invention, the term "promoter" refers to an expression control sequence that controls the initiation and rate of transcription of a gene or transgene. Promoters can be, for example, constitutive, inducible, repressive, or tissue-specific. Promoters may contain genetic elements that can bind to regulatory proteins and molecules such as RNA polymerases and transcription factors.

[0140] In this invention, when applied to promoters, the term "relative strength" refers to the percentage of the strength of the test promoter relative to the strength of a control promoter in the target strain or cell. Promoter strength can be determined using methods well known to those skilled in the art. For example, the promoter to be tested can be operatively linked to a nucleotide sequence encoding a fluorescent protein, such that the expression of the fluorescent protein is initiated by the promoter to be tested. In this case, the expression level of the fluorescent protein reflects the strength of the promoter to be tested.

[0141] In this invention, "operably linked" refers to the connection of nucleic acid sequences such that one sequence provides the function required for the linked sequences. In this invention, "operably linked" can mean linking a promoter to a sequence of interest, such that the transcription of the sequence of interest is controlled and regulated by the promoter. When the sequence of interest encodes a protein and its expression is desired, "operably linked" means that a promoter is linked to the sequence in a manner that enables efficient transcription and translation of the sequence.

[0142] In this invention, the term "frame-compliant linking" refers to linking two or more coding sequences in a manner that does not affect their translation into the original encoded amino acid sequences, thereby forming a fused coding sequence. Therefore, two or more coding sequences linked in a "frame-compliant linking" manner can encode fusion proteins.

[0143] In this invention, the terms "genome insertion" and "genome integration" are used interchangeably, referring to the insertion of a foreign DNA sequence or target gene fragment into the genome of a target strain.

[0144] In this invention, the terms "insertion site" and "integration site" are used interchangeably, referring to the target site in which a foreign DNA sequence or target gene fragment is inserted into the genome.

[0145] In some implementations, the target site for genome insertion can be an endogenous gene, and after the insertion of exogenous DNA, the original coding sequence of the endogenous gene is disrupted or replaced. In some implementations, the upstream and downstream sequences of the endogenous gene remain unchanged. In some implementations, the upstream and downstream sequences of the endogenous gene can be altered, for example, through homologous recombination.

[0146] In some implementations, the target site for genome insertion can be upstream or downstream of the endogenous gene, preferably within 1000 bp upstream or downstream. After the insertion of exogenous DNA, the original coding sequence of the endogenous gene remains unchanged.

[0147] In this invention, "genome overexpression" refers to the expression of a target gene in a target strain using the integration of exogenous DNA. In this case, the target gene fragment is inserted into the target site of the host cell genome and expressed. Methods for inserting exogenous DNA sequences or target gene fragments into the target site of the genome are well known to those skilled in the art. For example, exogenous DNA sequences or target gene fragments can be inserted into the target site of the genome using homologous recombination. Homologous recombination relies on the homology between DNA molecules. When applying homologous recombination, it is usually necessary to add upstream and downstream sequences homologous to the target integration site; these upstream and downstream sequences can also be referred to as homologous arms. In this invention, the homology required for homologous recombination is at least 10%. For example, if the length of the upstream or downstream sequence during homologous recombination is 1000 bp, then at least 100 bp of DNA needs to be identical to the other DNA in the homologous recombination.

[0148] In this invention, the term "expression cassette" refers to a promoter + gene fragment sequence + terminator, which is obtained by operatively linking the promoter, gene fragment sequence and terminator together.

[0149] Unless otherwise specified, the genes mentioned in the examples are all complete coding frames of genes, which can be obtained from NCBI. The promoter and terminator sequences mentioned can also be downloaded from NCBI. The specific sequence start position can be found from the primers in the primer table.

[0150] Example 1: Construction of a recombinant Saccharomyces cerevisiae producing decanpentol

[0151] The construction process of the recombinant Saccharomyces cerevisiae producing decanpentol is as follows: Figure 1 As shown.

[0152] (1) Construction of pBBS17-RsDDSA vector

[0153] Encoding type Rhodobulobacteria ( RhodobactersphaeroidesThe decanisopentene pyrophosphate synthase gene RsDDSA, derived from [source name], was first codon-optimized (sequence shown in SEQ ID NO:1) and then chemically synthesized by Shanghai Qingke Biotechnology Co., Ltd. The gene was then amplified by PCR using pre-designed primers RsDDSA-F / RsDDSA-R (sequences shown in SEQ ID NO:3 and SEQ ID NO:4). The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0154] Table 1 RsDDSA gene primers

[0155] .

[0156] The target plasmid pBBS17 (derived from patent CN116716196A, plasmid backbone pYES2, plasmid map shown) was processed using two enzymes, BamH1 and XhoI. Figure 2 The linearized vector fragment was subjected to double enzyme digestion, and the digestion products were detected by 1.0% agarose gel electrophoresis and purified by gel extraction. Then, the purified gene fragment was ligated to the linearized plasmid pBBS17 using Novizan's one-step cloning kit (37℃, 30 min). The ligation product was transformed into... E. coli DH5α was used to obtain the transformation product; the transformation product was plated on LB solid medium containing 100 mg / L ampicillin, and after obtaining single clones, it was cultured in shake flasks at 30℃. The plasmid was then extracted and sequenced for verification, thus obtaining the decanpentyl pyrophosphate synthase expression plasmid pBBS17-RsDDSA. Figure 3 ).

[0157] (2) The RsDDSA expression cassette was integrated into the starting brewer's yeast to obtain DOH01.

[0158] by Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, PCR was performed using primers GAL1-7-UF / GAL1-7-UR (sequences shown in SEQ ID NO:5 and SEQ ID NO:6) and GAL1-7-DF / GAL1-7-DR (sequences shown in SEQ ID NO:7 and SEQ ID NO:8) to obtain the PCR products of homologous arms GAL1-7-UP and GAL1-7-Dn. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0159] Table 2 Primers for GAL1-7 homologous arms

[0160] .

[0161] Using plasmid pBBS17-RsDDSA as a template, the PCR product of the RsDDSA expression cassette (pGAL1-RsDDSA-tCYC1) was obtained by PCR using primers GAL1-7-F / GAL1-7-R (sequences shown in SEQ ID NO:9 and SEQ ID NO:10). The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified using a clean-up kit. The purified products of homologous arms GAL1-7-UP and GAL1-7-Dn, as well as the purified product of the RsDDSA expression cassette, were then integrated into the gene using CRISPR / Cas9 gene editing technology. Saccharomyces cerevisiae The engineered yeast strain DOH01 was obtained from CEN.PK2-1C.

[0162] Table 3 Primers for RsDDSA Expression Cascade

[0163] .

[0164] The above-mentioned recombinant yeast strain DOH01 was inoculated into seed culture medium and cultured for 24 h. The seed liquid was then inoculated into 50 ml of fermentation medium at 1% (v / v) and cultured in a shaker at 30 °C for 72 h. The yield of decanterenol of the constructed engineered strain was 30.4 mg / L.

[0165] (3) Construction of pBBS17-RsDDSA-LPP1 vector

[0166] by Saccharomyces cerevisiae Using the CEN.PK2-1C genome as a template, and with the designed LPP1-F / LPP1-R (sequences shown in SEQ ID NO:17 and SEQ ID NO:18) as primers, phosphatidylphosphatase LPP1 was amplified by PCR. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0167] Table 4 LPP1 gene primers

[0168] .

[0169] The target plasmid pBBS17-RsDDSA was double-digested with NotI and BcuI enzymes. The digestion products were detected by 1.0% agarose gel electrophoresis, and the linearized vector fragment was purified by gel extraction. Then, the purified phosphatidyl phosphatase gene fragment was ligated into the linearized plasmid pBBS17-RsDDSA using Novizan's one-step cloning kit (37℃, 30 min). The ligation product was transformed into... E. coliDH5α was used to obtain the transformation product; the transformation product was plated on LB solid medium (containing a final concentration of 100 mg / L ampicillin), and after obtaining single clones, it was cultured in shake flasks at 37℃ and 220 rpm for 8-12 h. The plasmid was then extracted and sequenced for verification. If the verification was correct, the expression plasmid pBBS17-RsDDSA-LPP1 of the decanpentyl pyrophosphate synthase gene DDSA and the phosphatidyl phosphatase gene LPP1 was obtained. Figure 4 ).

[0170] (4) RsDDSA and LPP1 expression cassettes were integrated into Saccharomyces cerevisiae DOH01 to obtain DOH03.

[0171] by Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, PCR was performed using primers rDNA-UF / rDNA-UR (sequences shown in SEQ ID NO:23 and SEQ ID NO:24) and rDNA-DF / rDNA-DR (sequences shown in SEQ ID NO:25 and SEQ ID NO:26) to obtain PCR products of homologous arms rDNA-UP and rDNA-Dn. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0172] Table 5. Primers for rDNA homologous arms

[0173] .

[0174] Using plasmid pBBS17-RsDDSA-LPP1 as a template, PCR products of the RsDDSA and LPP1 expression cassette (tCYC1-RsDDSA-pGAL1-pGAL10-LPP1-tADH1) were obtained by PCR using primers rDNA-F / rDNA-R (sequences shown in SEQ ID NO:19 and SEQ ID NO:20). The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0175] Table 6 Primers for RsDDSA and LPP1 expression cassettes

[0176] .

[0177] by Saccharomyces cerevisiaeUsing S288C as a template, the PCR product of the LEU2 gene expression cassette (pLEU2-LEU2-tPGK1) was obtained by PCR using primers LEU2-F / LEU2-R (sequences shown in SEQ ID NO:21 and SEQ ID NO:22). The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified using a clean-up kit.

[0178] Table 7 LEU2 expression cassette primers

[0179] .

[0180] The purified products of homologous arms rDNA-UP and rDNA-Dn, the purified products of RsDDSA and LPP1 expression cassettes and LEU2 expression cassettes were integrated into the DOH01 multicopy site using endogenous homologous recombination of Saccharomyces cerevisiae to obtain the yeast engineered strain DOH03.

[0181] The above-mentioned recombinant yeast strain DOH03 was inoculated into seed culture medium and cultured for 24 h. The seed culture was then inoculated into 50 ml of fermentation medium at 1% (v / v) and cultured at 30℃ with shaking for 72 h. The yield of decanterenol in the constructed engineered strain was determined to be 13.06 ± 0.83 mg / L. Figure 7 (Table 15).

[0182] (5) Integrating the weak promoter PHXT1 into Saccharomyces cerevisiae DOH03 to obtain DOH04

[0183] by Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, the PCR product of promoter PHXT1 (sequence shown in SEQ ID NO:2) was obtained by PCR using primers PHXT1-F / PHXT1-R (sequences shown in SEQ ID NO:11 and SEQ ID NO:12). The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified using a clean-up kit.

[0184] Table 8. PHXT1 promoter primers

[0185] .

[0186] by Saccharomyces cerevisiaeUsing the CEN.PK2-1C or BY4741 genome as a template, PCR was performed using primers ProERG9-UF / ProERG9-UR (sequences shown in SEQ ID NO:13 and SEQ ID NO:14) and ProERG9-DF / ProERG9-DR (sequences shown in SEQ ID NO:15 and SEQ ID NO:16) to obtain PCR products of homologous arms ProERG9-UP and ProERG9-Dn. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0187] Table 9. Primers for ProERG9 homologous arms

[0188] .

[0189] Taking the construction method of yeast engineered strain DOH01 as an example, the promoter of the squalene synthase ERG9 gene was replaced with the weak promoter PHXT1 and integrated into the genome of yeast engineered strain DOH03 to obtain yeast engineered strain DOH04.

[0190] The above-mentioned recombinant yeast strain DOH04 was inoculated into seed culture medium and cultured for 24 h. The seed culture was then inoculated into 50 ml of fermentation medium at 1% (v / v) and cultured at 30℃ with shaking for 72 h. The yield of decanterenol in the constructed engineered strain was determined to be 20.13 ± 1.66 mg / L. Figure 7 (Table 15).

[0191] (6) Enhance the expression of endogenous MVA gene in yeast to obtain DOH07 from Saccharomyces cerevisiae DOH04.

[0192] Taking the construction method of plasmid pBBS17-RsDDSA-LPP1 as an example, Saccharomyces cerevisiae Using the CEN.PK2-1C or BY4741 genome as a template, expression plasmids for the five genes of the MVA pathway, ERG10, ERG13, tHMGR, ERG12 and ERG20, were constructed using plasmid pBBS17 as a vector (primer sequences are shown in SEQ ID NO:27-36). The constructed plasmids are pBBS17-ERG10-tHMGR, pBBS17-ERG12-ERG13 and pBBS17-ERG20-tHMGR, respectively.

[0193] Table 10 Primers for ERG12, ERG13, ERG10, tHMGR, and ERG20 genes

[0194] .

[0195] Taking the construction method of yeast engineered strain DOH03 as an example, the expression plasmids pBBS17-ERG10-tHMGR, pBBS17-ERG12-ERG13, and pBBS17-ERG20-tHMGR of the five genes in the MVA pathway were used as templates. PCR products of the corresponding expression cassettes of the five genes in the MVA pathway were obtained by PCR (primer sequences are shown in SEQ ID NO:37-38). The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit. The corresponding expression cassettes of the five genes in the MVA pathway obtained in this step are as follows:

[0196] (1)tCYC1-tHMGR-pGAL1-pGAL10-ERG10-tADH1

[0197] (2)tCYC1-ERG12-pGAL1-pGAL10-ERG13-tADH1

[0198] (3)tCYC1-tHMGR-pGAL1-pGAL10-ERG20-tADH1

[0199] Table 11 Primers for ERG10-tHMGR, ERG12-ERG13, and ERG20-tHMGR gene expression cassettes

[0200] .

[0201] Using homologous recombination technology of Saccharomyces cerevisiae, the corresponding expression cassettes of five genes in the MVA pathway were integrated into the 308a, 1114a, and 1021b sites of the genome of the yeast engineered strain DOH04 to obtain the yeast engineered strain DOH07.

[0202] The above-mentioned recombinant yeast strain DOH07 was inoculated into seed culture medium and cultured for 24 h. The seed culture was then inoculated into 50 ml of fermentation medium at 1% (v / v) and cultured at 30℃ with shaking for 72 h. The yield of decanterenol in the constructed engineered strain was determined to be 73.56 ± 2.05 mg / L. Figure 7 (Table 15).

[0203] (7) Integrating the lipid droplet-localized RsDDSA gene into Saccharomyces cerevisiae DOH07 to obtain DOH15

[0204] PLN1 is an endogenous gene in *Saccharomyces cerevisiae*, and its N-terminal signal peptide can guide gene localization to lipid droplets. Saccharomyces cerevisiaeUsing the CEN.PK2-1C or BY4741 genome as a template, the PCR product of the N-terminal localization signal peptide of the PLN1 gene (sequence shown in SEQ ID NO:41) was obtained by PCR using primers PLN1-F / PLN1-R (sequences shown in SEQ ID NO:39 and SEQ ID NO:40). The PCR product was detected by 1.0% agarose gel electrophoresis and the PLN1 signal peptide gene fragment was purified using a clean-up kit.

[0205] Using the synthesized gene RsDDSA (sequence shown in SEQ ID NO:1) as a template, and the designed primers RsDDSA-PLN1-F / RsDDSA-R (sequences shown in SEQ ID NO:42 and SEQ ID NO:4), the decanpentine pyrophosphate synthase gene was amplified by PCR. The PCR product was detected by 1.0% agarose gel electrophoresis and the RsDDSA gene fragment was purified using a clean-up kit.

[0206] Using the purified RsDDSA gene fragment and PLN1 signal peptide gene fragment as templates, and primers PLN1-F / RsDDSA-R (sequences shown in SEQ ID NO:39 and SEQ ID NO:4), the PLN1-RsDDSA fragment of the decanpentine pyrophosphate synthase gene, located in lipid droplets, was obtained by fusion PCR. Taking the construction method of pBBS17-RsDDSA plasmid as an example, an expression plasmid of RsDDSA located in lipid droplets was constructed using plasmid pBBS17 as a vector, which is pBBS17-PLN1-RsDDSA. Figure 5 ).

[0207] Table 12 Primers for PLN1 and RsDDSA genes

[0208] .

[0209] Taking the construction method of yeast engineered strain DOH01 as an example, the expression plasmid pBBS17-PLN1-RsDDSA, which locates the gene for the synthesis of decanopyrene pyrophosphate in lipid droplets, was used as a template. The PCR product of the PLN1-RsDDSA expression cassette (pGAL1-PLN1-RsDDSA-tCYC1) was obtained by PCR. The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by a clean-up kit.

[0210] Table 13 Primers for PLN1-RsDDSA Gene Expression Cassette

[0211] .

[0212] by Saccharomyces cerevisiaeUsing the CEN.PK2-1C or BY4741 genome as a template, PCR products of homologous arms 911b-UP and 911b-Dn were obtained by primers 911b-UF / 911b-UR (sequences shown in SEQ ID NO:43 and SEQ ID NO:44) and 911b-DF / 911b-DR (sequences shown in SEQ ID NO:45 and SEQ ID NO:46). The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit.

[0213] Table 14 911b homologous arm primers

[0214] .

[0215] Using homologous recombination technology of Saccharomyces cerevisiae, the PLN1-RsDDSA expression cassette was integrated into the 911b site of the genome of the yeast engineered strain DOH07 to obtain the yeast engineered strain DOH15.

[0216] The above-mentioned recombinant yeast strain DOH15 was inoculated into seed culture medium and cultured for 24 h. The seed culture was then inoculated into 50 ml of fermentation medium at 1% (v / v) and cultured at 30℃ with shaking for 72 h. The yield of decanterenol in the constructed engineered strain was determined to be 112.66 ± 5.37 mg / L. Figure 7 (Table 15).

[0217] Table 15. Yield of decanopenenol (mg / L) by engineered strains of Saccharomyces cerevisiae

[0218] .

[0219] This invention utilizes the lipid droplet localization signal peptide PLN1 linked to the co-expression of the RsDDSA gene. RsDDSA is located in lipid droplets and catalyzes the formation of decanopyridine pyrophosphate (DPP) from FPP and IPP, which is subsequently catalyzed by phosphatase to form decanopyrol. This invention employs this strategy to achieve the storage of the product decanopyrol within lipid droplets, resulting in a significant increase in the yield of the fermentation product decanopyrol.

[0220] The engineered Saccharomyces cerevisiae strain DOH15 constructed in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on May 22, 2025, with accession number CCTCC NO: M 20251155.

[0221] Fermentation broths of engineered strains DOH03 and DOH15 were analyzed by HPLC, and the results are as follows: Figure 6 As shown, compared with the standard decanoisopentenol, the target engineered bacteria DOH15 prepared in this invention ferments to obtain decanoisopentenol.

[0222] Example 2: Fermentation and product extraction and purification of recombinant Saccharomyces cerevisiae producing decanopentenol

[0223] The above-mentioned recombinant yeast engineered strain DOH15 was inoculated into seed culture medium and cultured for 24 h. The seed liquid was then inoculated into 3 L of fermentation medium at 10% (v / v). The culture was carried out in a 5 L fermenter at 30 °C using a fed-batch culture method for 120 h. The yield of the engineered strain was then tested, and the yield of decanterenol reached 1.04 g / L.

[0224] After solid-liquid separation and dehydration, the supernatant of the fermented liquid is adsorbed with resin and then eluted with a certain concentration of ethanol aqueous solution. The eluent is then distilled under reduced pressure to obtain crude decanpentol concentrate. The crude product is extracted with ethyl acetate, and the extract is then distilled under reduced pressure to recover ethyl acetate, thus obtaining the decanpentol product.

[0225] The seed culture medium formula is: 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0226] The fermentation medium formula is as follows: glucose 20 g / L, potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 2.9 g / L, yeast extract 10 g / L, and peptone 20 g / L.

[0227] Example 3: Screening and optimization process for locating signal peptide genes

[0228] The recombinant Saccharomyces cerevisiae genetically engineered strain prepared in this embodiment differs from strain DOH15 prepared in Example 1 only in the signal peptide gene used to locate RsDDSA in step (7). All other operations are the same as in Example 1. Specifically, the signal peptide genes used to locate RsDDSA are AAMB and Olesin, respectively, and the engineered strain was prepared according to the following method:

[0229] 1) Construction of the pBBS17-RsDDSA vector;

[0230] 2) The RsDDSA expression cassette was integrated into the starting brewer's yeast to obtain DOH01;

[0231] 3) Construction of the pBBS17-RsDDSA-LPP1 vector;

[0232] 4) The RsDDSA and LPP1 expression cassettes were integrated into Saccharomyces cerevisiae DOH01 to obtain DOH03;

[0233] 5) Integrating the weak promoter PHXT1 into Saccharomyces cerevisiae DOH03 yields DOH04;

[0234] 6) Enhance the expression of endogenous MVA genes in yeast to obtain DOH07 from Saccharomyces cerevisiae DOH04;

[0235] 7) The RsDDSA gene for lipid droplet localization was integrated into Saccharomyces cerevisiae DOH07 to obtain DOH15', in which the signal peptide genes for lipid droplet localization were AAMB and Olesin, respectively;

[0236] 7.1) AAMB is derived from Homo sapiens and Olesin signal peptide is derived from Zeamays. After codon optimization (sequences shown as SEQ ID NO:47 and SEQ ID NO:48, respectively), they were chemically synthesized by Shanghai Qingke Biotechnology Co., Ltd.

[0237] 7.2) Using RsDDSA (sequence shown in SEQ ID NO:1) as a template, primers were designed to amplify the decanisopentene pyrophosphate synthase gene by PCR. The PCR product was detected by 1.0% agarose gel electrophoresis and the RsDDSA gene fragment was purified by a clean-up kit.

[0238] 7.3) Using the RsDDSA gene fragment and the AAMB and Olesin signal peptide gene fragments as templates, the AAMB-RsDDSA fragment of the decanpentine pyrophosphate synthase gene located in lipid droplets was obtained by fusion PCR. The expression plasmid pBBS17-AAMB-RsDDSA located in lipid droplets was constructed using the plasmid pBBS17 as a vector. The expression plasmid pBBS17-Olesin-RsDDSA was obtained by the same method.

[0239] 7.4) Using pBBS17-AAMB-RsDDSA as a template, the PCR product of the AAMB-RsDDSA expression cassette (pGAL1-AAMB-RsDDSA-tCYC1) was obtained by PCR. The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by a clean-up kit. Similarly, the Olesin-RsDDSA expression cassette was obtained.

[0240] 7.5) Using homologous recombination technology of Saccharomyces cerevisiae, the AAMB-RsDDSA expression cassette was integrated into the 911b site of the genome of the yeast engineered strain DOH07 to obtain the yeast engineered strain DOH15. 1 The Olesin-RsDDSA expression cassette was integrated into the 911b site of the genome of the engineered yeast strain DOH07 to obtain the engineered yeast strain DOH15. 2 The RsDDSA expression cassette (a lipid droplet-free localization control) was integrated into the 911b site of the genome of the engineered yeast strain DOH07 to obtain the engineered yeast strain DOH15. 3 .

[0241] Take the above-mentioned recombinant yeast strain DOH15 1DOH15 2 DOH15 3 The seed culture was inoculated into seed culture medium and cultured for 24 h. The seed culture was then taken and inoculated into 50 ml of fermentation medium at 1% (v / v). The culture was carried out at 30 °C with shaking for 72 h. The yields of decanterenol of the constructed engineered strain were 102.1 mg / L, 85.4 mg / L, and 74.5 mg / L, respectively.

[0242] Compared with the lipid droplet localization signal peptide PLN1, when the signal peptide gene was replaced with AAMB or Olesin, the ability of the resulting engineered strain to produce decanopentenol decreased, while direct expression of RsDDSA did not increase the yield. This indicates that, compared with direct expression of RsDDSA, different lipid droplet signal peptides can increase the yield of RsDDSA gene. However, the effects of different signal peptides on RsDDSA expression are different, with signal peptide PLN1 showing the best effect in localizing RsDDSA to lipid droplets.

[0243] The above specific embodiments are merely illustrative of the content of the present invention and do not represent a limitation of the content of the present invention. Those skilled in the art will realize that the specific structure of the present invention can have other variations.

Claims

1. A recombinant brewer's yeast, characterized in that, Compared to the original Saccharomyces cerevisiae strain, the recombinant Saccharomyces cerevisiae has a combination of the characteristics shown in 1)-5): 1) Expressing the RsDDSA gene, a decanpentene pyrophosphate synthase derived from Rhodococcus globulus; 2) Overexpression of the endogenous dephosphatase LPP1 gene; 3) Inhibit the squalene synthase ERG9 gene; 4) Overexpression of MVA pathway genes ERG10, ERG13, tHMGR, ERG12, and ERG20; 5) Expressing the RsDDSA gene, a lipid droplet localization gene derived from Rhodococcus globulus; The RsDDSA gene has the nucleotide sequence shown in SEQ ID NO:1; The NCBI number for the endogenous dephosphatase LPP1 gene of the Saccharomyces cerevisiae is NM_001180811.3; The squalene synthase ERG9 gene is expressed by a weak promoter pHXT1, the nucleotide sequence of which is shown in SEQ ID NO:2; The NCBI numbers for the MVA pathway genes ERG10 are Gene ID: 856079, ERG13 is Gene ID: 854913, tHMGR is Gene ID: 854900, ERG12 is Gene ID: 855248, and ERG20 is Gene ID: 853272. The lipid droplet-localized RsDDSA gene is a fusion gene of the N-terminal signal peptide gene of the PLN1 gene and RsDDSA, and the nucleotide sequence of the N-terminal signal peptide of the PLN1 gene is shown in SEQ ID NO:

41.

2. The recombinant Saccharomyces cerevisiae according to claim 1, characterized in that, The RsDDSA gene is integrated into the GAL1-7 gene site and rDNA site in the Saccharomyces cerevisiae genome to express the RsDDSA gene via the promoter pGAL1. The LPP1 gene is integrated into the rDNA site in the Saccharomyces cerevisiae genome to express the endogenous dephosphatase LPP1 gene of Saccharomyces cerevisiae via the promoter pGAL10. ERG10 and tHMGR genes were integrated into the 308a site of the Saccharomyces cerevisiae genome, ERG12 and ERG13 genes were integrated into the 1114a site of the Saccharomyces cerevisiae genome, and ERG20 and tHMGR genes were integrated into the 1021b site of the Saccharomyces cerevisiae genome. The N-terminal signal peptide gene of the PLN1 gene is integrated with the RsDDSA fusion gene into the 911b site of the Saccharomyces cerevisiae genome to express the lipid droplet-localized RsDDSA gene via the promoter pGAL1.

3. The recombinant Saccharomyces cerevisiae according to any one of claims 1-2, characterized in that, The starting strain of the brewing yeast is brewing yeast CEN.PK2-1D.

4. The recombinant Saccharomyces cerevisiae according to claim 3, characterized in that, The recombinant Saccharomyces cerevisiae is Saccharomyces cerevisiae (Sac Saccharomyces cerevisiae DOH15 was deposited at the China Center for Type Culture Collection (CCTCC) on May 22, 2025, with accession number CCTCC NO: M 20251155.

5. A method for constructing the recombinant Saccharomyces cerevisiae producing decanoisopentenol as described in claim 1, characterized in that, This includes the following modifications to the starting brewer's yeast strain: S1) expresses the RsDDSA gene, a decanpentene pyrophosphate synthase derived from Rhodophyta globulinii; and S2) Overexpression of the endogenous dephosphatase LPP1 gene in Saccharomyces cerevisiae; and S3) Downregulated the transcriptional level of squalene synthase ERG9 in the cell matrix of Saccharomyces cerevisiae; and S4) Overexpression of MVA pathway genes ERG10, ERG13, tHMGR, ERG12, and ERG20; and S5) expresses the RsDDSA gene, a lipid droplet-localized cyclophosphamide-derived decanopyrene pyrophosphate synthase.

6. The method according to claim 5, characterized in that, The method for constructing a recombinant Saccharomyces cerevisiae that produces decanopentenol includes the following steps: 1) Construct the RsDDSA gene expression cassette and integrate it into the GAL1-7 site of the starting yeast strain through homologous recombination to obtain yeast strain DOH01; 2) Construct RsDDSA and LPP1 gene expression cassettes, and integrate the RsDDSA and LPP1 gene expression cassettes into the rDNA site of the chromosome of yeast strain DOH01 through homologous recombination mechanism to obtain yeast strain DOH03; 3) Based on yeast strain DOH03, the ERG9 promoter was replaced with the pHXT1 promoter to obtain yeast strain DOH04; 4) Construct ERG10 and tHMGR gene expression cassettes, ERG12 and ERG13 gene expression cassettes and ERG20 and tHMGR gene expression cassettes, and integrate the gene expression cassettes into the 308a, 1114a and 1021b sites of the yeast strain DOH04 chromosome through homologous recombination mechanism to obtain yeast strain DOH07; 5) The N-terminal signal peptide gene of the PLN1 gene was fused with the RsDDSA gene to obtain the fusion gene PLN1-RsDDSA. The PLN1-RsDDSA gene expression cassette was constructed and integrated into the 911b site of the chromosome of yeast strain DOH07 through homologous recombination to obtain yeast strain DOH15.

7. The use of the recombinant Saccharomyces cerevisiae according to any one of claims 1-4 or the recombinant Saccharomyces cerevisiae constructed by the method according to any one of claims 5-6 in at least one of the following: 1) Application in the fermentation production of decanoisopentenol; 2) Application in the genetic breeding of microorganisms that produce decanpentol; 3) In the production of Coenzyme Q 10 Applications in [the field].

8. A method for producing decanoisopentenol by fermentation, characterized in that, The method includes the step of culturing the recombinant yeast as described in any one of claims 1-4 or constructing the recombinant yeast by the method described in any one of claims 5-6.

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