Recombinant yarrowia lipolytica engineering bacterium with high yield of sclareol and construction of recombinant yarrowia lipolytica engineering bacterium

Abstract

The invention provides a recombinant yarrowia lipolytica engineering bacterium for high-yield sclareol and construction of the recombinant yarrowia lipolytica engineering bacterium, and belongs to the technical field of sclareol biosynthesis. Yarrowia lipolytica Po1f delta Ku70 is used as a chassis bacterium, and the following genetic background of the yarrowia lipolytica Po1f delta Ku70 is modified: an SsLPPS gene and an SsSCS gene from sclarela sclareana are expressed; the method comprises the following steps: overexpressing an endogenous HMG1 gene and an IDI1 gene, positioning the endogenous HMG1 gene and the IDI1 gene in mitochondria, overexpressing a SsGGPPS gene from clary sage, overexpressing a fusion gene formed by SsLPPS and SsSCS, and overexpressing a fusion gene formed by SsGGPPS and SsLPPS. According to the invention, after the endogenous MVA pathway key gene is subjected to mitochondrial zoning, the heterologous key gene is over-expressed, the expression of the heterologous gene is regulated and controlled by enzyme fusion and promoter engineering, and the Leu defect gene is supplemented, so that a multi-dimensional metabolic engineering scheme is formed, and a new normal form is provided for efficient production of sclareol.

Description

Technical Field

[0001] The present invention relates to the technical field of sclareol biosynthesis, and in particular to a recombinant Yarrowia lipolytica engineered bacterium capable of producing high sclareol yield, a construction method and an application thereof. Background Art

[0002] Sclareol is a diterpenoid compound with the chemical formula C 20 H 36 O2 is primarily used as a fragrance ingredient in cosmetics and perfumes, as well as a food flavoring. It is also a raw material for the chemical synthesis of ambroxan, an ambergris substitute. Therefore, sclareol has high economic value. Furthermore, sclareol exhibits anti-tumor and anti-inflammatory activities. Currently, sclareol is primarily extracted from plants, but plants grow slowly and are susceptible to environmental influences, pests and diseases, which can affect the quality and yield of the extracted sclareol.

[0003] Using microbial cell factories to produce high-value-added compounds from plant sources is of great significance in solving the problems of insufficient plant raw materials and low chemical synthesis yields. With the development of metabolic engineering and synthetic biology, many terpenoid compounds have been efficiently produced in microbial cell factories.

[0004] Yarrowia lipolytica, a "Generally Recognized as Safe" strain, has attracted close attention from researchers due to its abundant supply of precursors and cofactors such as acetyl-CoA, its lack of the Crabtree effect, and its tolerance to high osmotic pressure. In a study titled "Constructing a green oleaginous yeast cell factory for sustainable production of the plant-derived diterpenoid sclareol," published in the journal Green Chemistry in February 2024, the engineered strain of Yarrowia lipolytica produced 817.7 mg / L of sclareol in a shake flask fermentation for 120 h. In a 5-L fermentor, fed-batch fermentation for 240 h yielded 12.9 g / L of sclareol, but the yield was relatively low, at 0.054 g / L / h. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present invention further transforms Yarrowia lipolytica and successfully constructs a Yarrowia lipolytica cell factory that can efficiently produce sclareol, solving the technical problem of low efficiency of existing Yarrowia lipolytica engineered bacteria in producing sclareol.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a method for constructing a recombinant Yarrowia lipolytica engineered bacterium with high sclareol production, namely, using the Yarrowia lipolytica Po1fΔ Ku70 For the bottom plate bacteria, the following treatments are carried out in the bottom plate bacteria: Expressing the gene encoding lysostigmine diol pyrophosphate synthase from Clary Sage ( SsLPPS gene) and the gene encoding sclareol synthase ( SsSCS Gene); Overexpression of the gene encoding endogenous HMG-CoA reductase ( HMG1 gene) and the gene encoding isopentenyl pyrophosphate isomerase ( IDI1 genes), and localizes both to mitochondria; Overexpression of the gene encoding geranylgeranyl pyrophosphate synthase from Sclarea sclarea ( SsGGPPS Gene); Overexpression SsLPPS and SsSCS The fusion gene formed and SsGGPPS and SsLPPS The formed fusion gene.

[0007] Preferably, in the above construction scheme, each gene is integrated into the Yarrowia lipolytica genome, and the integration sites include but are not limited to IntF, IntA2, IntD10, IntF17, IntD4, LIG4, IntA1, etc.

[0008] Preferably, in the above construction method, the connecting peptide used in gene fusion expression is any one of EEEK, TPTP, GGGGS, GSG, (EEK)2, (TPTP)2, (GGGGS)2, and (GSG)2. The type of connecting peptide has a significant effect on the ability of the resulting engineered bacteria to produce sclareol. As shown by the experimental data of the present invention, the gene SsLPPS and SsSCS When expressing by fusion, TPTP is the best linker peptide.

[0009] In the above construction method, the promoter used for each gene expression has an impact on the ability of the resulting engineered bacteria to produce sclareol, especially for the expression of the two fusion genes. As shown by the experimental data of the present invention, SsLPPS and SsSCS The overexpression of the fusion gene was performed using P hp4d promoter, the effect was significantly better than that of P TEFin promoter; and for SsGGPPS and SsLPPS Overexpression of the fusion gene formed, P GPDin The promoter is due to P TEFinTherefore, in order to obtain better technical effects, those skilled in the art may try to further optimize the promoter used for expression of each gene.

[0010] In the above construction method, the mitochondrial localization signal peptide MLS can be used to convert the MVA pathway into HMG1 、 IDI1 The enzyme encoded by the gene is localized in the mitochondria.

[0011] Preferably, in the above construction method, the bottom plate bacteria also includes the following processing: Overexpression of Saccharomyces cerevisiae ScHMG1 genes; and / or, Cover Leu Defective gene.

[0012] In a second aspect, the present invention provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of sclareol, specifically obtained by the construction method described above. For example, in some specific embodiments of the present invention, the following recombinant Yarrowia lipolytica engineered strains that produce sclareol are provided: Engineered strain SCL01, with Po1fΔ Ku70 As the starting strain, the expression of Clary Sage-derived SsLPPS Genes and SsSCS Gene; The engineered strain SCL02, based on the engineered strain SCL01, overexpresses endogenous HMG1 Genes and IDI1 The gene is located in mitochondria; The engineered strain SCL03, based on the engineered strain SCL02, overexpresses the SsGGPPS Gene; The engineered strain SCL04, based on the engineered strain SCL03, overexpresses SsLPPS and SsSCS The fusion gene formed; The engineered strain SCL05, based on the engineered strain SCL03, overexpresses SsLPPS and SsSCS The fusion gene was formed, and the promoter used for overexpression of the fusion gene was optimized to P hp4d ; The engineered strain SCL06, based on the engineered strain SCL05, overexpresses SsGGPPS and SsLPPS The fusion gene formed; The engineered strain SCL07, based on the engineered strain SCL05, overexpresses SsGGPPS and SsLPPS The fusion gene was formed, and the promoter used for overexpression of the fusion gene was optimized to P GPDin ; The engineered strain SCL08, based on the engineered strain SCL07, overexpresses the ScHMG1 Gene; The engineered strain SCL09 is based on the engineered strain SCL08 and supplemented with Leu Defective gene.

[0013] Experimental data showed that after 120 hours of shake flask fermentation, the production of sclareol by the engineered strain SCL04 reached 976.2 mg / L. The sclareol production capacity of each engineered strain gradually increased thereafter. After 120 hours of shake flask fermentation, the production of sclareol by the engineered strain SCL09 reached 2895.4 mg / L, far exceeding the existing technology.

[0014] In a third aspect, the present invention provides a method for producing sclareol, using the high-sclareol-producing recombinant Yarrowia lipolytica constructed according to the present invention as a fermentation strain to produce sclareol. Specifically, the method comprises the following steps: inoculating a seed solution of the fermentation strain into a fermentation medium containing glucose and performing fed-batch fermentation; wherein, during the fermentation process, the pH is maintained at 5-6, and after a period of fermentation, 10%-20% (v / v) isopropyl myristate is added, and the glucose concentration is controlled to 1-3 g / L in a fed-batch manner. For example, in one embodiment of the present invention, the engineered strain SCL09 achieved a sclareol yield of 30.7 g / L after 192 hours of fed-batch fermentation, demonstrating that this method is highly suitable for the industrial production of sclareol.

[0015] Fourthly, the recombinant Yarrowia lipolytica strains of the present invention that produce high levels of sclareol can be used as a base cell to prepare engineered strains for the synthesis of ambroxan. Sclareol is a raw material for the chemical synthesis of ambroxan, an ambergris substitute. Therefore, those skilled in the art can further engineer the engineered strains provided by the present invention to produce a Yarrowia lipolytica cell factory for the production of ambroxan.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention expresses a gene encoding lisdendiolate pyrophosphate synthase in Yarrowia lipolytica SsLPPS , genes encoding sclareol synthase SsSCS Achieve de novo synthesis of sclareol and localize HMG1 and IDI1 to mitochondria and overexpress them SsGGPPS Gene, overexpression SsLPPS and SsSCS The fusion gene formed SsGGPPS and SsLPPS The fusion gene formed and complemented LeuBy removing a defective gene, a recombinant Yarrowia lipolytica engineered strain capable of high-sclareol production was constructed. By regulating the expression of heterologous genes in the sclareol metabolic pathway, efficient sclareol synthesis was achieved. Furthermore, the production of sclareol using the recombinant Yarrowia lipolytica engineered strain of the present invention is environmentally friendly, pollution-free, high-yield, and low-cost, with significant industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0018] Figure 1 The roadmap for synthesizing sclareol by the engineered strain of Yarrowia lipolytica in the examples; Figure 2 The sclareol production and OD of each recombinant Yarrowia lipolytica strain constructed in the example 600 Comparison result chart of values; Figure 3 In the embodiment SsLPPS and SsSCS Sclareol production and OD when different linker peptides were used in gene fusion 600 Comparison result chart of values; Figure 4 This is a graph showing the change in sclareol production when the engineered strain SCL01 was fermented in a 5 L fermenter for 192 h; Figure 5 The maps of plasmids pUC19-IntF-LPPS-SCS (left) and pUC19-IntA2-MLS-HMG1-MLS-IDI (right) in the examples are shown; Figure 6 The maps of plasmids pUC19-IntD10-SsGGPPS (left) and pUC19-IntF17-LPPS-TPTP-SCS in the examples are shown; Figure 7 The maps of the plasmids pUC19-IntF17-hp4d-LPPS-TPTP-SCS (left) and pUC19-IntD4-SsGGPPS-EEEK-SsLPPS in the examples are shown; Figure 8 The maps of the plasmids pUC19-IntD4-GPDin-SsGGPPS-EEEK-SsLPPS (left) and pUC19-LIG4-ScHMG1 in the examples are shown; Figure 9: is a map of the plasmid pUC19-IntA1-Leu in the examples. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains; the terms used herein are for the purpose of describing specific implementations only and are not intended to limit the present invention; the term "include" and any variations thereof herein are intended to cover non-exclusive inclusions.

[0021] Modifying Yarrowia lipolytica to construct a Yarrowia lipolytica cell factory for sclareol production is a promising approach to addressing market demand for sclareol. However, existing Yarrowia lipolytica engineered strains (e.g., patent applications CN119351238A, CN117264792A, and CN114989997) still exhibit low sclareol production. To address this technical issue, the present invention provides a high-sclareol-producing recombinant Yarrowia lipolytica engineered strain and a method for its construction. The resulting engineered strain achieves a maximum sclareol yield of 30.7 g / L after 192 hours of fed-batch fermentation, and the construction method is also simpler to operate.

[0022] Reference Figure 1 The method for constructing a recombinant Yarrowia lipolytica engineered bacterium with high sclareol production provided in an embodiment of the present invention comprises the following steps: Yarrowia lipolytica Po1fΔ Ku70 For the bottom plate bacteria, the following treatments are carried out in the bottom plate bacteria: Expressing the origin of Clary Sage SsLPPS Genes and SsSCS Gene; Overexpression of endogenous HMG1 Genes and IDI1 gene and localize it to mitochondria; Overexpression of Clary Sclarea-derived SsGGPPS Gene; Overexpression SsLPPS and SsSCS The fusion gene formed and SsGGPPS and SsLPPS The formed fusion gene.

[0023] Furthermore, in some embodiments, in order to further increase the yield of sclareol, the bottom plate bacteria also includes the following treatment: Overexpression of Saccharomyces cerevisiaeScHMG1 Gene; and / or, complement Leu Defective gene.

[0024] Gene SsLPPS 、 SsSCS 、 HMG1 、 IDI1 、 SsGGPPS 、 ScHMG1, Leu The nucleotide sequences of the genes are available for query in NCBI, and their GeneBank accession numbers are JQ478434.1, JN133922.1, UTQH01000001.1, XM_504974.3, MK442922.1, Z46373.1, and CP061014.1, respectively. In order to achieve the expression of each of the exogenous genes in Yarrowia lipolytica, the codons of the genes derived from Sclarea sclarea were optimized in the present invention, and the optimized SsLPPS 、 SsSCS 、 SsGGPPS The nucleotide sequences are SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively.

[0025] In the above construction scheme, each gene is integrated into the Yarrowia lipolytica genome. The specific integration sites used in the examples are IntF, IntA2, IntD10, IntF17, IntD4, LIG4, and IntA1.

[0026] Further, in some embodiments, the promoter used for inserting gene expression is selected from P TEFin 、P FABin 、P hp4d 、P GPDin ; The terminator used is selected from T PEX20 、T CYC .

[0027] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0028] The specific composition of each culture medium used in the following examples is as follows: YPD liquid medium: 20 g / L tryptone, 20 g / L glucose, 10 g / L yeast extract, sterilized at 115°C for 30 min; corresponding solid medium is supplemented with 20 g / L agar; YPD-60 liquid medium: 20 g / L tryptone, 60 g / L glucose, 10 g / L yeast extract, sterilized at 115°C for 30 min; YNB-Ura / YNB-Leu solid medium: 20 g / L glucose, 6.7 g / L amino-free yeast nitrogen base (YNB), 0.1 g / L leucine (Leu) / uracil (Ura), 20 g / L Agar, sterilized at 115°C for 30 min.

[0029] In the following examples, the Uniclone One-Step Seamless Cloning Kit was purchased from Beijing Jinsha Biotechnology Co., Ltd. The method for use was as follows: based on the number of base pairs in the fragment and the recovered concentration, the volume of each fragment was calculated according to the instructions. Water, fragment, vector, and 2 × Uniclone Seamless Cloning Mix were added to a PCR tube in that order. The tube was mixed thoroughly and incubated in a 50°C water bath for 30 min.

[0030] In the following examples, yeast transformation was performed using the Frozen-EZ Yeast Transformation II kit (ZymoResearch Corporation), and the operating procedures are described in the instructions.

[0031] In the following examples, gene integration was performed using the Cre-loxP system via homologous recombination. To ensure continued use of the URA3 selection tag in experiments, the URA3 tag needed to be recovered. This recovery method involved transforming the strain pRRQ2 containing the Cre protein to obtain a sclareol-producing engineered strain, plating the strain on YNB-Leu plates and culturing at 30°C for 2 days. Transformants from the YNB-Leu plates were streaked onto YNB-Ura and YPD plates, respectively. Transformants that did not grow on YNB-Ura but grew on YPD indicated that the URA3 tag had been recovered. The nucleotide sequence of the LoxR / LoxP-containing URA3 used in the examples is shown in SEQ ID No. 4.

[0032] Example 1 This example provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of claryol. The construction method comprises the following steps: (1) Construct a sclareol biosynthesis pathway in Yarrowia lipolytica.

[0033] Po1fΔ Ku70 The strain was the starting strain, which expressed the SsLPPS and SsSCSGene implementation includes the following operations: Synthesized at Jinkairui Bioengineering Co., Ltd. SsLPPS Genes and SsSCS gene, and used it as a template to amplify SsLPPS Genes and SsSCS Gene fragment. Ku70 The genome was used as a template to amplify the promoter P TEFin 、P FABin and terminator T PEX20 、T CYC The promoter, gene, terminator and pUC19 plasmid backbone were connected using the Uniclone OneStep Seamless Cloning Kit to construct the expression cassette plasmid pUC19-P TEFin -LPPS-T PEX20 and pUC19-P FABin -SCS-T CYC .

[0034] In order to SsLPPS Genes and SsSCS The gene was integrated into the yeast chromosome IntF site, and about 1000 bp upstream and downstream of the IntF site were selected as upstream and downstream homology arms. Ku70 The genome was used as a template to amplify the upstream and downstream homology arms of the IntF locus and the URA3 fragment with LoxR / LoxP. The pUC19-IntF-URA3 plasmid was constructed using the Uniclone One Step Seamless Cloning Kit. The primers for amplifying the upstream homology arm of IntF were IntF-UP-F / IntF-UP-R (sequences shown in SEQ ID Nos. 5 and 6, respectively), and the primers for amplifying the downstream homology arm of IntF were IntF-DOWN-F / IntF-DOWN-R (sequences shown in SEQ ID Nos. 7 and 8, respectively).

[0035] plasmid pUC19-P TEFin -LPPS-T PEX20 、pUC19-P FABin -SCS-T CYC and pUC19-IntF-URA3 as templates to amplify the gene expression cassette P TEFin -LPPS-T PEX20 and P FABin -SCS-T CYC and the pUC19-IntF-URA3 fragment with homology arms, and P TEFin -LPPS-T PEX20 and PFABin -SCS-T CYC The gene expression cassette and vector pUC19-IntF-URA3 fragment were cloned in one step using the Uniclone One Step Seamless Cloning Kit to construct the plasmid pUC19-IntF-LPPS-SCS ( Figure 5 center left).

[0036] Using the plasmid pUC19-IntF-LPPS-SCS as a template, the fragment containing the homology arms and the gene expression cassette was amplified and recovered using primers IntF-UP-F / IntF-DOWN-R. The recovered fragment was transformed into the Po1f-ΔKu70 strain using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for 4 days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL01.

[0037] A single colony of the engineered strain SCL01 was inoculated into a 10 mL centrifuge tube containing 1 mL YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed solution was inoculated into a 100 mL conical flask containing 15 mL YPD medium and cultured for 24 h with an inoculation volume of 10% (v / v). The second-level seed solution was inoculated into a 250 mL conical flask containing 50 mL YPD-60 medium with an inoculation volume of 5% (v / v). 10% (v / v) isopropyl myristate was added and fermented under the same conditions for 120 h. The yield of sclareol was 4.6 mg / L. SsLPPS and SsSCS gene, achieving de novo synthesis of sclareol in Yarrowia lipolytica.

[0038] (2) Overexpression of key genes in the MVA pathway.

[0039] Based on the engineered strain SCL01, this study inserted the key genes of the MVA pathway into the HMG1 、 IDI1 The encoded enzyme is localized in mitochondria. The engineered strain SCL02 was constructed, and the specific operations are as follows: Using the Po1f-ΔKu70 genome as a template, primers were used to amplify HMG1 、 IDI1 Gene fragment; Specifically, the sequence of the mitochondrial localization signal peptide MLS is designed in the primer to amplify HMG1 The primers for the gene are HMG1-F / HMG1-R (sequences are shown in SEQ ID No.9-10 respectively), and the amplification IDI1The primers for the gene are IDI1-F / IDI1-R (sequences are shown in SEQ ID No.11-12 respectively). TEFin -LPPS-T PEX20 and pUC19-P FABin -SCS-T CYC As a template, the pUC19 backbone with promoter and terminator was amplified. The expression cassette plasmid pUC19-P was constructed by ligation using the Uniclone One Step Seamless Cloning Kit. TEFin -MLS-HMG1-T PEX20 and pUC19-P FABin -MLS-IDI1-T CYC .

[0040] In order to gene HMG1 and genes IDI1 The plasmid pUC19-IntA2-URA3 was constructed by integrating the gene at the IntA2 locus on the yeast chromosome according to the method in step (1). The primers for amplifying the upstream homology arm of IntA2 were IntA2-UP-F / IntA2-UP-R (sequences shown in SEQ ID No. 13-14, respectively), and the primers for amplifying the downstream homology arm of IntA2 were IntA2-DOWN-F / IntA2-DOWN-R (sequences shown in SEQ ID No. 15-16, respectively).

[0041] plasmid pUC19-P TEFin -MLS-HMG1-T PEX20 、pUC19-P FABin -MLS-IDI1-T CYC and pUC19-IntA2-URA3 as templates, respectively, to amplify the gene expression cassette P TEFin -MLS-HMG1-T PEX20 、P FABin -MLS-IDI1-T CYC and the pUC19-IntA2-URA3 fragment with homology arms, and the plasmid pUC19-IntA2-MLS-HMG1-MLS-IDI was constructed by one-step cloning. Figure 5 center right).

[0042] Using the plasmid pUC19-IntA2-MLS-HMG1-MLS-IDI as a template, primers IntA2-UP-F and IntA2-DOWN-R were used to amplify and recover a fragment containing homology arms and the gene expression cassette. The recovered fragment was transformed into the engineered strain SCL01, which had been modified to contain the URA3 tag, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for four days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL02.

[0043] A single colony of the engineered strain SCL02 was inoculated into a 10 mL centrifuge tube containing 1 mL YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed solution was inoculated into a 100 mL conical flask containing 15 mL YPD medium and cultured for 24 h with an inoculation volume of 10% (v / v). The second-level seed solution was inoculated into a 250 mL conical flask containing 50 mL YPD-60 medium with an inoculation volume of 5% (v / v). 10% (v / v) isopropyl myristate was added and fermented under the same conditions for 120 h. The yield of sclareol was 16.7 mg / L. It can be seen from the fermentation results that the MVA pathway is involved in the production of sclareol. HMG1 、 IDI1 After the gene was localized in the mitochondria, the sclareol production was 3.6 times that of the SCL01 strain.

[0044] (3) Overexpression of key genes SsGGPPS .

[0045] Based on the engineered strain SCL02, the geranylgeranyl pyrophosphate synthase gene from Clary Sage was overexpressed. SsGGPPS , the engineered strain SCL03 was constructed, and the specific operations were as follows: Synthesized at Jinkairui Bioengineering Co., Ltd. SsGGPPS Gene, using it as a template for amplification SsGGPPS Gene fragment. TEFin -LPPS-T PEX20 As a template, the pUC19 backbone with promoter and terminator was amplified. The expression cassette plasmid pUC19-P was constructed by one-step cloning kit. TEFin -SsGGPPS-T PEX20 .

[0046] In order to SsGGPPSThe gene was integrated into the IntD10 locus on the yeast chromosome, and the plasmid pUC19-IntD10-URA3 with homology arms was constructed according to step (1). The primers for amplifying the upstream homology arm of IntD10 were IntD10-UP-F / IntD10-UP-R (sequences shown in SEQ ID No. 17-18, respectively), and the primers for amplifying the downstream homology arm of IntD10 were IntD10-DOWN-F / IntD10-DOWN-R (sequences shown in SEQ ID No. 19-20, respectively).

[0047] plasmid pUC19-P TEFin -SsGGPPS-T PEX20 and pUC19-IntD10-URA3 as templates to amplify the gene expression cassette P TEFin -SsGGPPS-T PEX20 The plasmid pUC19-IntD10-SsGGPPS was constructed by one-step cloning with the vector pUC19-IntD10-URA3 fragment. Figure 6 center left).

[0048] Using the plasmid pUC19-IntD10-SsGGPPS as a template, primers IntD10-UP-F and IntD10-DOWN-R were used to amplify and recover the fragment containing the homology arms and the gene expression cassette. The recovered fragment was transformed into the engineered strain SCL02, which had the URA3 tag removed, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After incubation at 30°C for 4 days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL03.

[0049] A single colony of the engineered strain SCL03 was inoculated into a 10 mL centrifuge tube containing 1 mL YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed solution was inoculated into a 100 mL conical flask containing 15 mL YPD medium and cultured for 24 h at an inoculation volume of 10% (v / v). The second-level seed solution was inoculated into a 250 mL conical flask containing 50 mL YPD-60 medium at an inoculation volume of 5% (v / v). 10% (v / v) isopropyl myristate was added and fermented under the same conditions for 120 h. The yield of sclareol was 141.3 mg / L. As can be seen from the fermentation results, the overexpressed gene SsGGPPS Afterwards, the recombinant strain SCL03 increased the sclareol production by 8.5 times compared with the strain SCL02.

[0050] (4) Fusion expression SsLPPS and SsSCSGene.

[0051] On the basis of the engineered strain SCL03, the expression of Clary Sage-derived SsLPPS Genes and SsSCS gene, and in this case TPTP is used as the connecting peptide. The specific operations are as follows: The connecting peptide TPTP sequence was designed into the primers and amplified to obtain SsLPPS gene and with a connecting peptide TPTP SsSCS Gene fragment. TEFin -LPPS-T PEX20 As a template, the pUC19 backbone with promoter and terminator was amplified. Finally, the expression cassette plasmid pUC19-P was constructed by one-step cloning. TEFin -LPPS-TPTP-SCS-T PEX20 .

[0052] In order to SsLPPS and SsSCS The fusion gene was integrated into the yeast chromosome IntF17 site, and the homology arm plasmid pUC19-IntF17-URA3 was constructed according to step (1). The primers used to amplify the upstream homology arm of IntF17 were IntF17-UP-F / IntF17-UP-R (sequences shown in SEQ ID No. 21-22, respectively), and the primers used to amplify the downstream homology arm of IntF17 were IntF17-DOWN-F / IntF17-DOWN-R (sequences shown in SEQ ID No. 23-24, respectively).

[0053] plasmid pUC19-P TEFin -LPPS-TPTP-SCS-T PEX20 and pUC19-IntF17-URA3 as templates to amplify P TEFin -LPPS-TPTP-SCS-T PEX20 The expression cassette and vector pUC19-IntF17-URA3 fragment were then cloned into the plasmid pUC19-IntF17-LPPS-TPTP-SCS ( Figure 6 center right).

[0054] Using the plasmid pUC19-IntF17-LPPS-TPTP-SCS as a template, primers IntF17-UP-F and IntF17-DOWN-R were used to amplify and recover the fragment containing the homology arms and the gene expression cassette. The recovered fragment was transformed into the engineered strain SCL03, which had the URA3 tag removed, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for four days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL04.

[0055] A single colony of the engineered strain SCL04 was inoculated into a 10 mL centrifuge tube containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed solution was inoculated into a 100 mL conical flask containing 15 mL of YPD medium and cultured for 24 h at an inoculum volume of 10% (v / v). The second-level seed solution was inoculated into a 250 mL conical flask containing 50 mL of YPD-60 medium at an inoculum volume of 5% (v / v). 10% (v / v) isopropyl myristate was added and fermented under the same conditions for 120 h. The yield of sclareol was 976.2 mg / L. As can be seen from the fermentation results, SsLPPS and SsSCS Gene fusion expression significantly increased the production of sclareol. The sclareol production of the recombinant strain SCL04 was 6.9 times that of the strain SCL03.

[0056] In addition, SsLPPS Genes and SsSCS After the connecting peptide between genes is replaced, the ability of the recombinant bacteria to produce sclareol will change, such as Figure 3 shown.

[0057] Example 2 This example provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of claryol. The construction method comprises the following steps: Steps (1), (2), and (3) are the same as those in Example 1.

[0058] (4) Different from Example 1, this example uses the promoter P hp4d control LPPS - TPTP - SCS Fusion gene expression, that is, promoter P TEFin Replace with P hp4d The specific operations are as follows: The hp4d promoter and pUC19-IntF17-LPPS-TPTP-SCS backbone were amplified and the expression cassette plasmid pUC19-IntF17-hp4d-LPPS-TPTP-SCS was constructed by one-step cloning ( Figure 7center left).

[0059] Using the plasmid pUC19-IntF17-hp4d-LPPS-TPTP-SCS as a template, primers IntF17-UP-F and IntF17-DOWN-R were used to amplify and recover a fragment containing homology arms and the gene expression cassette. The recovered fragment was transformed into the engineered strain SCL03, which had the URA3 tag recovered, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for 4 days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL05.

[0060] A single colony of the engineered strain SCL05 was inoculated into a 10-mL centrifuge tube containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The primary seed solution was inoculated into a 100-mL Erlenmeyer flask containing 15 mL of YPD medium and cultured for 24 h at a 10% (v / v) inoculum. The secondary seed solution was inoculated into a 250-mL Erlenmeyer flask containing 50 mL of YPD-60 medium at a 5% (v / v) inoculum. 10% (v / v) isopropyl myristate was added. After 120 h of fermentation under the same conditions, the sclareol yield in the SCL05 strain was 1578.3 mg / L. The fermentation results indicate that, using the hp4d promoter to control the expression of the LPPS-TPTP-SCS fusion gene, the sclareol yield in the SCL05 strain increased by 61.7% compared to the SCL04 strain.

[0061] Example 3 This example provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of claryol. The construction method comprises the following steps: (1) The engineered strain SCL05 was obtained according to the method in Example 2.

[0062] (2) Fusion expression SsGGPPS and SsLPPS Gene.

[0063] On the basis of the engineered strain SCL05, the expression of SsGGPPS Genes and SsLPPS gene, and in this case, EEEK is used as the connecting peptide. The specific operations are as follows: The connecting peptide EEEK sequence was designed into the primers and amplified SsGGPPS gene and the linker peptide EEEK SsLPPS Gene fragment. TEFin -LPPS-T PEX20As a template, amplify the pUC19 backbone with promoter and terminator. Finally, one-step cloning is used to construct the expression cassette plasmid pUC19-P TEFin -SsGGPPS-EEEK-SsLPPS-T PEX20 .

[0064] In order to SsGGPPS and SsLPPS The fusion gene was integrated into the yeast chromosome at the IntD4 locus, and the homology arm plasmid pUC19-IntD4-URA3 was constructed according to Example 1. The primers used to amplify the upstream homology arm of IntD4 were IntD4-UP-F / IntD4-UP-R (sequences shown in SEQ ID Nos. 25 and 26, respectively), and the primers used to amplify the downstream homology arm of IntD4 were IntD4-DOWN-F / IntD4-DOWN-R (sequences shown in SEQ ID Nos. 27 and 28, respectively).

[0065] plasmid pUC19-P TEFin -SsGGPPS-EEEK-SsLPPS-T PEX20 and pUC19-IntD4-URA3 as templates to amplify P TEFin -SsGGPPS-EEEK-SsLPPS-T PEX20 The expression cassette was combined with the vector pUC19-IntD4-URA3 fragment, and then the plasmid pUC19-IntD4-SsGGPPS-EEEK-SsLPPS was constructed by one-step cloning. Figure 7 center right).

[0066] Using the plasmid pUC19-IntD4-SsGGPPS-EEEK-SsLPPS as a template, primers IntD4-UP-F and IntD4-DOWN-R were used to amplify and recover the fragment containing the homology arms and the gene expression cassette. The recovered fragment was then transformed into the engineered strain SCL05, which had the URA3 tag recovered, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for 4 days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL06.

[0067] A single colony of the engineered strain SCL06 was inoculated into a 10 mL centrifuge tube containing 1 mL YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed solution was inoculated into a 100 mL conical flask containing 15 mL YPD medium and cultured for 24 h. The inoculation volume was 10% (v / v). The second-level seed solution was inoculated into a 250 mL conical flask containing 50 mL YPD-60 medium. The inoculation volume was 5% (v / v). 10% (v / v) isopropyl myristate was added. After fermentation for 120 h under the same conditions, the yield of sclareol was 1914.4 mg / L. As can be seen from the fermentation results, the expression of sclareol by fusion with the connecting peptide EEEK was SsGGPPS and SsLPPS gene, the sclareol production of SCL06 strain was increased by 21.3% compared with that of SCL05 strain.

[0068] Example 4 This example provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of claryol. The construction method comprises the following steps: (1) The engineered strain SCL05 was obtained according to the method in Example 2.

[0069] (2) Different from Example 3, this example uses the promoter P GPDin control SsGGPPS - EEEK - SsLPPS The specific steps for fusion gene expression are as follows: P GPDin The promoter and pUC19-IntD4-SsGGPPS-EEEK-SsLPPS backbone were constructed by one-step cloning to construct the expression cassette plasmid pUC19-IntD4-GPDin-SsGGPPS-EEEK-SsLPPS ( Figure 8 center left).

[0070] Using the plasmid pUC19-IntD4-GPDin-SsGGPPS-EEEK-SsLPPS as a template, primers IntD4-UP-F and IntD4-DOWN-R were used to amplify and recover the fragment containing the homology arms and the gene expression cassette. The recovered fragment was transformed into the engineered strain SCL05, which had the URA3 tag recovered, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for 4 days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL07.

[0071] A single colony of the engineered strain SCL07 was inoculated into a 10 mL centrifuge tube containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed liquid was inoculated into a 100 mL Erlenmeyer flask containing 15 mL of YPD medium and cultured for 24 h at an inoculum size of 10% (v / v). The second-level seed liquid was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD-60 medium at an inoculum size of 5% (v / v). 10% (v / v) isopropyl myristate was added. After fermentation under the same conditions for 120 h, the sclareol yield was 2364.7 mg / L, which was 23.5% higher than that of the SCL06 strain and 49.8% higher than that of the SCL05 strain.

[0072] Example 5 This example provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of claryol. The construction method comprises the following steps: (1) The engineered strain SCL07 was obtained according to the method in Example 4.

[0073] (2) Overexpression HMG1 Gene.

[0074] Based on the engineered strain SCL07, this study further overexpressed the Saccharomyces cerevisiae-derived [[ID=I41]]ScHMG1 Gene, the specific operations are as follows: Amplification ScHMG1 pUC19-P TEFin -LPPS-T PEX20 As a template, the pUC19 backbone with promoter and terminator was amplified, and then the expression cassette plasmid pUC19-P was constructed by one-step cloning. TEFin -ScHMG1-T PEX20 .

[0075] In order to ScHMG1 The gene was integrated into the yeast chromosome at the LIG4 locus, and the homology arm plasmid pUC19-LIG4-URA3 was constructed according to Example 1. The primers for amplifying the upstream homology arm of LIG4 were LIG4-UP-F / LIG4-UP-R (sequences shown in SEQ ID Nos. 29 and 30, respectively), and the primers for amplifying the downstream homology arm of LIG4 were LIG4-DOWN-F / LIG4-DOWN-R (sequences shown in SEQ ID Nos. 31 and 32, respectively).

[0076] plasmid pUC19-P TEFin -ScHMG1-T PEX20 and pUC19-LIG4-URA3 as templates to amplify P TEFin -ScHMG1-TPEX20 The expression cassette was combined with the vector pUC19-LIG4-URA3 fragment, and then the plasmid pUC19-LIG4-ScHMG1 was constructed by one-step cloning ( Figure 8 center right).

[0077] Using the plasmid pUC19-LIG4-ScHMG1 as a template, primers LIG4-UP-F and LIG4-DOWN-R were used to amplify and recover the fragment containing the homology arms and the gene expression cassette. The recovered fragment was then transformed into the engineered strain SCL07, which had been modified to contain the URA3 tag, using the Frozen-EZ Yeast Transformation II Kit and plated onto YNB-Ura plates. After culturing in a 30°C incubator for 4 days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL08.

[0078] A single colony of the engineered strain SCL08 was inoculated into a 10 mL centrifuge tube containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The first-level seed solution was inoculated into a 100 mL conical flask containing 15 mL of YPD medium and cultured for 24 h with an inoculation volume of 10% (v / v). The second-level seed solution was inoculated into a 250 mL conical flask containing 50 mL of YPD-60 medium with an inoculation volume of 5% (v / v). 10% (v / v) isopropyl myristate was added and fermented under the same conditions for 120 h. The yield of sclareol was 2678.1 mg / L. As can be seen from the fermentation results, the overexpression of the Saccharomyces cerevisiae-derived ScHMG1 gene, the sclareol production of the SCL08 strain was increased by 13.3% compared with that of the SCL07 strain.

[0079] Example 6 This example provides a recombinant Yarrowia lipolytica engineered strain that produces high levels of claryol. The construction method comprises the following steps: (1) The engineered strain SCL08 was obtained according to the method in Example 5.

[0080] (2) Complement the leucine-deficient gene.

[0081] Amplification Leu The gene fragment and the pUC19 backbone with promoter and terminator were cloned into the expression cassette plasmid pUC19-P by one-step cloning. TEFin -Leu-T PEX20 .

[0082] In order to LeuThe gene was integrated into the yeast chromosome at the IntA1 locus, and the homology arm plasmid pUC19-IntA1 was constructed according to Example 1. The primers for amplifying the upstream homology arm of IntA1 were IntA1-UP-F / IntA1-UP-R (sequences shown in SEQ ID Nos. 33-34, respectively), and the primers for amplifying the downstream homology arm of IntA1 were IntA1-DOWN-F / IntA1-DOWN-R (sequences shown in SEQ ID Nos. 35-36, respectively).

[0083] plasmid pUC19-P TEFin -Leu-T PEX20 and pUC19-IntA1 as templates to amplify Leu The gene expression cassette and the plasmid pUC19-IntA1 fragment with homology arms were constructed by one-step cloning of the expression cassette plasmid pUC19-IntA1-Leu ( Figure 9 ).

[0084] Using the plasmid pUC19-IntA1-Leu as a template, primers IntA1-UP-F and IntA1-DOWN-R were used to amplify and recover a fragment containing homology arms and the gene expression cassette. The recovered fragment was then transformed into the engineered strain SCL08 using the Frozen-EZ Yeast Transformation II Kit and plated onto the leucine-deficient selection plate YNB-Leu. After culturing in a 30°C incubator for four days, positive transformants were selected and verified by PCR to obtain the recombinant engineered strain SCL09.

[0085] A single colony of the engineered strain SCL09 was inoculated into a 10-mL centrifuge tube containing 1 mL of YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h. The primary seed solution was inoculated into a 100-mL Erlenmeyer flask containing 15 mL of YPD medium and cultured for 24 h at a 10% (v / v) inoculum. The secondary seed solution was inoculated into a 250-mL Erlenmeyer flask containing 50 mL of YPD-60 medium at a 5% (v / v) inoculum. 10% (v / v) isopropyl myristate was added. After 120 h of fermentation under the same conditions, the sclareol yield of the recombinant strain SCL09 was 2895.4 mg / L. The fermentation results indicate that, after complementation of the leucine-deficient gene, the sclareol yield of the recombinant strain SCL09 increased by 8.1% compared to that of the strain SCL08.

[0086] Example 7 This example uses the engineered strain SCL09 as an example. In this example, fed-batch fermentation is used to produce sclareol. The specific operation is as follows: The engineered strain SCL09 single clone was inoculated into a 10 mL centrifuge tube containing 1 mL YPD medium and cultured in a shaker at 250 rpm and 30°C for 24 h; 0.5 mL of the first-level seed solution was inoculated into a 250 mL conical flask containing 50 mL YPD medium and cultured for 24 h; the second-level seed solution was inoculated into a 5 L fermenter containing 1.5 L YPD-60 medium to make the initial OD 600 The fermentation temperature was 30°C, the pH was adjusted to 5.5 with ammonia, the dissolved oxygen was controlled at 20%, and the flow rate was 1 vvm. After 24 h of fermentation, 20% (v / v) isopropyl myristate was added. After the glucose was depleted, 800 g / L of glucose was added to maintain the glucose level at approximately 1 g / L. After 192 h of fermentation, the yield of sclareol reached 30.7 g / L (see Figure 4 ).

[0087] In summary, the present invention successfully constructed a recombinant Yarrowia lipolytica engineered strain capable of high-yielding sclareol by regulating the expression of heterologous genes in the sclareol metabolic pathway, achieving efficient synthesis of sclareol and being suitable for the industrial production of sclareol.

[0088] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A method for constructing a recombinant Yarrowia lipolytica engineered bacterium with high sclareol production, characterized in that: Yarrowia lipolytica Po1fΔ Ku70 For the bottom plate bacteria, the following treatments are carried out in the bottom plate bacteria: Expressing the origin of Clary Sage SsLPPS Genes and SsSCS Gene; Overexpression of endogenous HMG1 Genes and IDI1 gene and localize it to mitochondria; Overexpression of Clary Sclarea-derived SsGGPPS Gene; Overexpression SsLPPS and SsSCS The fusion gene formed and SsGGPPS and SsLPPS The formed fusion gene.

2. The construction method according to claim 1, characterized in that In the fusion gene, the connecting peptide is any one of EEEK, TPTP, GGGGS, GSG, (EEEK)2, (TPTP)2, (GGGGS)2, and (GSG)2.

3. The construction method according to claim 1, characterized in that described SsLPPS and SsSCS The overexpression of the fusion gene was performed using P hp4d promoter.

4. The construction method according to claim 1, characterized in that described SsGGPPS and SsLPPS The overexpression of the fusion gene was performed using P GPDin promoter.

5. The construction method according to claim 1, characterized in that The bottom plate bacteria also includes the following treatment: overexpression of Saccharomyces cerevisiae ScHMG1 Gene.

6. The construction method according to claim 1 or 5, characterized in that: The bottom plate bacteria also includes the following treatments: Leu Defective gene.

7. A recombinant Yarrowia lipolytica engineered bacterium capable of producing high levels of sclareol prepared according to any one of claims 1 to 6.

8. A method for producing sclareol, characterized in that: The recombinant Yarrowia lipolytica with high sclareol production according to claim 7 is used as a fermentation strain to produce sclareol.

9. The method according to claim 8, characterized in that The seed liquid of the fermentation strain was inoculated into a fermentation medium containing glucose. During the fermentation process, the pH value was controlled to be maintained at 5-6, and 10%-20% (v / v) isopropyl myristate was added. The glucose concentration was controlled to 1-3 g / L by batch feeding.

10. Use of the recombinant Yarrowia lipolytica engineered bacterium capable of producing high sclareol yield according to claim 7 in the preparation of engineered bacteria for synthesizing ambroxan.

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