Construction method and application of high-yield salidroside recombinant yarrowia lipolytica

Abstract

The invention provides a construction method and application of recombinant yarrowia lipolytica with high yield of salidroside, and belongs to the field of bioengineering. The recombinant yarrowia lipolytica with high yield of salidroside is named as a yarrowia lipolytica XJ-SAL strain, and after the recombinant yarrowia lipolytica XJ-SAL strain is subjected to fed-batch fermentation, the salidroside can be synthesized at a relatively high level. The construction method of the recombinant yarrowia lipolytica is easy to operate, and efficient synthesis of salidroside can be achieved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a method for constructing and applying a recombinant lipophilic yeast that produces high levels of rhodioloside. Background Technology

[0002] Salidroside, also known as tyrosol 8-O-β-D-glucopyranoside (2-(4-hydroxyphenyl)-ethyl-β-D-glucopyranoside), is a glycoside compound with important pharmacological activity, mainly found in the genus Rhodiola (Rhodiola rosea). Rhodiola Rhodioloside is found in the underground organs of plants. Its chemical characteristic is the formation of a glycosidic bond between the 8-hydroxyl group of tyrosol and β-D-glucose, a unique chemical structure that endows it with rich pharmacological activities. Studies have shown that rhodioloside not only possesses powerful antioxidant, anti-fatigue, and anti-aging activities, but also exhibits anticancer, anti-inflammatory, and neuroprotective activities, making it widely used in cosmetics, health products, and the medical field.

[0003] Traditionally, rhodioloside is mainly obtained through direct extraction from Rhodiola rosea plants, using methods such as hot reflux extraction, Soxhlet extraction, ultrasound-assisted extraction, microwave-assisted extraction, and infrared-assisted extraction. In addition, synthesizing rhodioloside through culturing plant cells or plant callus tissue is a novel approach currently being explored. However, this method faces challenges such as large planting areas, long growth cycles, and climate influences, resulting in low economic efficiency. Many existing chemical synthesis processes exist; for example, using tyrosol or tyrosol analogs as raw materials and utilizing catalysts such as heavy metal salts to catalyze the substrate glycosylation to generate rhodioloside. Shi et al. pioneered a kilogram-scale chemical synthesis process consisting of two chemical steps: first, an acetylation reaction to protect the phenolic hydroxyl group of tyrosol, followed by an O-glycosylation reaction with tetraacetyl bromide glucose under the catalysis of silver carbonate to synthesize rhodioloside. The overall yield was 72%, and the purity was higher than 98% (Shi, TY, Chen, H., Jing, LL, Liu, XY, Sun, XL, Jiang, R. (2011) Development of a kilogram-scale synthesis of salidroside and its analogs[J]. Synthetic Communications (41, 2594-2600). Although chemical synthesis methods offer higher yields and purity, they require various chemical reagents as protective agents or catalysts, using tyrosol or its analogues as substrates. Therefore, these methods are costly, unsafe, and not suitable for scale-up. Furthermore, because rhodioloside is an important raw material for food, pharmaceuticals, and health products, products obtained through chemical methods are not widely popular.

[0004] Rhodioloside is primarily synthesized in nature by plants of the Rhodiola genus, and its metabolic pathway has been elucidated, making microbial synthesis of rhodioloside possible. Microbial fermentation, with its short growth cycle, ability to operate around the clock, and advantages of low cost, mild conditions, and minimal environmental pollution, is currently a hot research topic. However, existing technologies lack microorganisms capable of efficiently producing rhodioloside. Yersinia lipolytica, as an unconventional oil-producing yeast, possesses a natural Ehrlich pathway and a high-throughput pentose phosphate pathway, making it highly suitable for industrial production. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing and applying a recombinant lipophilic yeast that produces high levels of rhodioloside, addressing the shortcomings of the prior art.

[0006] To address the aforementioned technical problems, this invention discloses a method for constructing and applying a recombinant lipophilic yeast that produces high levels of rhodioloside. The specific technical solution is as follows: In a first aspect, the present invention provides a method for constructing a recombinant *Yersinia lipolytica* strain that produces high levels of rhodioloside, wherein the recombinant *Yersinia lipolytica* strain expresses a glycosyltransferase gene. UGT phenylpyruvate decarboxylase gene ARO10 transketonease gene TKL DAHP synthase mutant gene AroG S180F and bifunctional branched acid mutase / prephenyl acid dehydrogenase mutant gene TyrA M53I / A354V Simultaneously, the prebenzoic acid dehydratase gene was knocked out. PHA2 and anthranilate synthase gene TRP3 ; wherein, the aforementioned UGT Derived from Arabidopsis thaliana Arabidopsis thaliana , is AtUGT85A1. Wherein, the aforementioned UGT The nucleotide sequence is shown in SEQ ID NO.3.

[0007] Among them, the ARO10 Derived from brewer's yeast Saccharomyces cerevisiae Or Yeast extract Yarrowia lipolytica Preferably, the ARO10 Derived from brewer's yeast; the aforementioned TKL Derived from Yersinia lipolytica Yarrowia lipolytica The aforementioned AroG S180F and TyrA M53I / A354V All were derived from Escherichia coli Escherichia coli In some embodiments of the present invention, the yeast is derived from brewer's yeast.Saccharomyces cerevisiae of ARO10 The nucleotide sequence is shown in SEQ ID NO.6, which is derived from Saccharomyces cerevisiae. ARO10 The mutant gene; derived from Yersinia lipolyticis. Y. lipolytica of ARO10 The gene number is YALI1_D08884g; it is derived from Yersinia lipolytica. Y. lipolytica of TKL The gene number is YALI1_E07744g; it originates from Escherichia coli. E.coli of AroG S180F and TyrA M53I / A354V The nucleotide sequences are shown in SEQ ID NO. 4~5, respectively. PHA2 The gene number is YALI1_B22549g; the aforementioned TRP3 The gene number is YALI1_D13963g.

[0008] The recombinant Yersinia lipophila expressed 1-3 copies of the yeast. ARO10 Two are preferred.

[0009] Among them, the recombinant Yersinia lipolyticis simultaneously contains the pyruvate kinase gene ( YlPYK The natural promoter P) PYK Truncation, the truncated P PYK The nucleotide sequence is shown in SEQ ID NO.7. For details on the truncation method, please refer to Chinese Patent CN120082582A.

[0010] Preferably, the present invention provides a method for constructing recombinant Yersinia lipolytica, wherein the recombinant Yersinia lipolytica expresses Arabidopsis thaliana. A.thaliana Source of glycosyltransferase gene AtUGT85A1 Escherichia coli E.coli DAHP synthase mutant gene from which the gene originated EcAROG S180F Escherichia coli E.coli Bifunctional branched acid mutase / prephenyl acid dehydrogenase mutant gene EcTYRA M53I / A354V and 2 copies of brewing yeast S.cerevisiae Phenylpyruvate decarboxylase mutant gene of origin ScARO10 I335E Overexpression of endogenous transketolase genes in Yeastia lipophila YlTKL Simultaneously, the prebenzoic acid dehydratase gene was knocked out. PHA2 and anthranilate synthase gene TRP3 and the natural promoter P of the pyruvate kinase gene PYKCut off to P PYK-jd .in, AtUGT85A1 , EcAROG S180F , EcTYRA M53I / A354V , ScARO10 I335E and P PYK-jd The nucleotide sequences are shown in SEQ ID NO.3-7, respectively. PHA2 and TRP3 The gene numbers are YALI1_B22549g and YALI1_D13963g, respectively.

[0011] The recombinant Yersinia lipophila mentioned above is a starter strain that has been knocked out. KU70 Yeast lipolysis Yarrowia lipolytica Po1f. Po1f was purchased from the American Culture Collection (ATCC) under the number ATCC MYA-2613.

[0012] The expressed or overexpressed gene is integrated into *Yarrowia lipophila*. Yarrowia lipolytica On the genome, the integration site is selected from any one or more of the following sites: A08 site, 26S rDNA site, IntA site, IntB site, IntC site, IntD site, IntE site, IntF site, lip1 site, SCP2 site, or YlSCD site.

[0013] Preferably, the present invention also provides a method for constructing the recombinant Yersinia lipolytica, wherein the target gene is integrated into the yeast genome in the form of a gene expression cassette. Specifically, it includes the following steps: (1) Construction of the target gene expression cassette: The promoter, target gene, and terminator are amplified to obtain promoter gene fragments, target gene fragments, and terminator gene fragments, respectively, which are then ligated into a plasmid vector to obtain a recombinant plasmid vector containing the target gene expression cassette; the promoter is the P promoter of Yersinia lipophila. TEF P hp4d P TEFin P FBA P FBAin P POX2 P POT1 P GPD or P EXP Any one of the following; the terminator is the terminator T of *Yarrowia lipolyticis*. xpr2t T mig1t T lip2t T cyc1t T pex3t T pex10t or T pex20tAny one of the following; the target gene includes any one of the following: glycosyltransferase gene, DAHP synthase mutant gene, bifunctional branched acid mutase / prephenyl acid dehydrogenase mutant gene, phenylpyruvate decarboxylase gene, phenylpyruvate decarboxylase mutant gene or transketolase gene.

[0014] (2) The recombinant plasmid vector containing the target gene expression cassette was introduced into Yersinia lipolytica so that the gene expression cassette was integrated into the Yersinia lipolytica genome; (3) Construct the prebenzoic acid dehydratase gene knockout cassette, the anthranilic acid synthase gene knockout cassette, and the pyruvate kinase gene promoter truncated expression cassette, and integrate them into the Yersinia lipolytica genome obtained in step (2). The construction method of the pyruvate kinase gene promoter truncated expression cassette is detailed in Chinese Patent CN120082582A.

[0015] Secondly, the present invention provides a recombinant Yersinia lipophila constructed by the construction method described in the first aspect.

[0016] Thirdly, the present invention provides the application of the recombinant *Yersinia lipolytica* described in the second aspect in the fermentation production of rhodioloside. Specifically, the recombinant *Yersinia lipolytica* is inoculated into a fermentation medium and fermented at 28-32°C for 4-7 days to obtain rhodioloside. Preferably, the fermentation is carried out at 28-30°C for 5-6 days. The fermentation medium comprises 20-100 g / L glucose, 5-20 g / L yeast extract, and 10-30 g / L peptone.

[0017] Beneficial effects: The recombinant lipophilic Yersinia constructed in this invention Yarrowia lipolytica XJ-SAL expresses the glycosyltransferase gene, the DAHP synthase mutant gene, the bifunctional branched acid mutase / prebenzoic acid dehydrogenase mutant gene, and the phenylpyruvate decarboxylase mutant gene; overexpresses the transketase gene; and knocks out the prebenzoic acid dehydratase gene and the anthranilic acid synthase gene. It also expresses the pyruvate kinase encoding gene from the endogenous yeast *Yarrowia lipolytica*. YlPYK The natural promoter was truncated to 300 bp. Experiments showed that the recombinant Yersinia lipolytica could produce 21528.9 mg / L of rhodioloside after fed-batch fermentation, achieving efficient synthesis of rhodioloside in Yersinia lipolytica. Attached Figure Description

[0018] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0019] Figure 1This is a metabolic diagram of rhodioloside synthesis provided in an embodiment of the present invention. Ac-CoA: Acetyl-CoA; CHA: Cranialized acid; DAHP: 3-deoxy-D-arabinohepenulose-7-phosphate; DHQ: 3-dehydroquinine; DHS: 3-dehydroshikimate; EPSP: 5-enolpyruvate-shikimate-3-phosphate; E4P: Erythrose-4-phosphate; F1,6BP: Fructose-1,6-bisphosphate; F6P: Fructose-6-phosphate; Glucose; G1P: Glucose-1-phosphate; G3P: Glyceraldehyde-3-phosphate; G6P: Glucose-6-phosphate; PEP: Phosphoene Alcoholic pyruvate; PPA: prephenylacetic acid; PYR: pyruvate; RU5P: ribulose-5-phosphate; R5P: ribose-5-phosphate; Salidroside: rhodioloside; SHIK: shikimic acid; S3P: shikimic acid-3-phosphate; S7P: sedoheptulose-7-phosphate; Tyrosol: tyrosol; UDP-Glucose: UDP-glucose; X5P: xylulose-5-phosphate; 4HPAA: 4-hydroxyphenylacetaldehyde; 4HPP: 4-hydroxyphenylpyruvate; AtUGT85A1: glycosyltransferase; Ec AroG S180F : DAHP synthase mutant; Ec TyrA M53I / A354V : Bifunctional branched acid mutase / prebenzoic acid dehydrogenase mutant; ScARO10 I335E : Phenylacetate decarboxylase mutant; Yl Pgm2: Phosphoglucose mutase UDP; Yl Pha2: Prephenylacetic acid dehydratase; Yl Tkl: transketolase; Yl Trp3: o-aminobenzoic acid synthase; YlUgp1: uridine dihydrotransferase.

[0020] Figure 2 For recombinant plasmid pUC-A08- EcAROG S180F The plasmid map.

[0021] Figure 3 The recombinant plasmid pUC-HUH-IntA- AtUGT85A1 The plasmid map.

[0022] Figure 4 Comparison of the content of rhodioloside (SAL) produced by fermentation of recombinant strains 1~13 (YL-1~YL-13). Detailed Implementation

[0023] The present invention will be further described below through specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0024] In the following examples, the lipophilic yeast (Yersinia lipophila) Yarrowia lipolytica Po1f, YPD plate formulation containing 5-fluoroorotic acid, SD-Leu screening medium formulation, SD-Ura screening medium formulation, A08 site integrative plasmid, IntA site integrative plasmid, IntB site integrative plasmid, IntC site integrative plasmid, IntE site integrative plasmid, plasmid pUC57-HisG-Ura-HisG, plasmid pUC57-Leu, promoter P TEFin Nucleotide sequence and terminator T xpr2t Nucleotide sequences are detailed in Chinese patent CN120082582A. Terminator T lip2t and T cyc1t For the nucleotide sequence details, please refer to Chinese Patent CN118421490A.

[0025] The biosynthetic pathway of rhodioloside described in this invention is as follows: Figure 1 As shown.

[0026] Example 1 (I) Preparation of target gene According to information from Rhodiola rosea on the NCBI website ( Rhodiola rosea ) glycosyltransferase gene (GenBank accession number: MF674558.1), Rhodiola rosea ( Rhodiola sachalinensis ) glycosyltransferase gene (GenBank accession number: EU567325.1) and Arabidopsis thaliana ( Arabidopsis thaliana The nucleotide sequence of the glycosyltransferase gene (GenBank accession number: AT1G22400 / NP_173656.1) from [source missing] was obtained, and after codon optimization, the optimized glycosyltransferase gene was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. [The remaining text appears to be incomplete and requires further context.] RrU8GT33 (SEQ ID NO.1) RsUGT72B14 (SEQ ID NO.2) and AtUGT85A1 (SEQ ID NO.3), and inserted into plasmid pUC57 (purchased from GenScript), to obtain plasmids pUC57-RrU8GT33, pUC57-RsUGT72B14 and pUC57-AtUGT85A1.

[0027] According to information from E. coli provided by NCBI ( E.coliThe nucleotide sequence of the DAHP synthase mutant gene (GenBank accession number: EET3970042.1) was obtained from [source name missing], and after codon optimization, the optimized DAHP synthase mutant gene was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. EcAROG S180F (SEQ ID NO.4), and inserted into plasmid pUC57 (purchased from GenScript), to obtain plasmid pUC57- EcAROG S180F .

[0028] According to information from E. coli provided by NCBI ( E. coli The nucleotide sequence of the bifunctional cladistic acid mutase / prephenyl acid dehydrogenase mutant gene (GenBank accession number: CP084207.1) was obtained, and after codon optimization, the optimized cladistic acid mutase gene was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. EcTYRA M53I / A354V (SEQ ID NO. 5), and inserted into plasmid pUC57 to obtain plasmid pUC57- EcTYRA M53I / A354V .

[0029] According to information from brewer's yeast provided on NCBI ( S.cerevisiae The nucleotide sequence of the phenylpyruvate decarboxylase mutant gene (GenBank accession number: EF059264.1) from [source missing] was codon-optimized, and the optimized phenylpyruvate decarboxylase mutant gene was synthesized by Suzhou Genewiz Biotechnology Co., Ltd. ScARO10 I335E (SEQ ID NO. 6), and inserted into plasmid pUC57 (purchased from GenScript), to obtain plasmid pUC57- ScARO10 I335E .

[0030] by Y. lipolytica Po1f Using Δku70 genomic DNA as a template, the transketolase gene was obtained by PCR amplification using specific primers. YlTKL (Gene number YALI1_E07744g) and phenylpyruvate decarboxylase gene YlARO10 (Gene number YALI1_D08884g).

[0031] (II) Construction of recombinant plasmids 1. Recombinant plasmid pUC-A08- EcAROG S180F Construction Recombinant plasmid pUC-A08- EcAROG S180FUsing the A08 site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of A08. EcAROG S180F Expression Box (P) TEFin - EcAROG S180F -T xpr2t For details, see [link / details]. Figure 2 .

[0032] by Y. lipolytica Using Po1f Δku70 genomic DNA as a template, primers in Table 1 were used to amplify the following sequences: EcAROG S180F Expression box promoter P TEFin and Termination T xpr2t Using plasmid pUC57- EcAROG S180F Using the template, amplification was performed using the primers in Table 1. EcAROG S180F Genes. Fragments were purified and recovered using the TaKaRa MiniBEST DNA Fragment Purification Kit (purchased from Shanghai Bioscientific and Technological Co., Ltd.).

[0033] The A08 site integration plasmid was processed using a restriction endonuclease from NEB. SnaB I. After enzyme digestion, the linearized A08 site integrated plasmid was recovered by agarose gel electrophoresis.

[0034] The linearized A08 site integration plasmid and the plasmid constructed in this embodiment were used. EcAROG S180F The components in the gene expression cassette (promoter P) TEFin ,Gene EcAROG S180F and Termination T xpr2t Using the ClonExpress MultiS One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., one-step cloning was achieved. EcAROG S180F The gene expression cassette was inserted between the upstream and downstream homologous arms of the A08 site integration plasmid to obtain a circular recombinant vector.

[0035] The circular recombinant vector was transformed into E. coli DH5α competent cells, and the cells were screened using ampicillin-resistant LB plates and verified by colony PCR and sequencing to obtain the positive recombinant plasmid pUC-A08-. EcAROG S180F .

[0036] Using restriction endonucleases from NEB Not Ⅰ For plasmid pUC-A08- EcAROG S180FAfter enzyme digestion, linearized pUC-A08 was recovered by agarose gel electrophoresis. EcAROG S180F Plasmids are used for yeast transformation.

[0037] Table 1 Recombinant plasmid pUC-A08- EcAROG S180F Primers used in construction

[0038] 2. Recombinant plasmid pUC-HUH-IntA- RrU8GT33 Construction Recombinant plasmid pUC-HUH-IntA- RrU8GT33 Using the IntA site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntA. RrU8GT33 Gene expression cassette (P TEFin - RrU8GT33 -T lip2t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similarly, the difference lies in the skeleton, and the expression box is replaced with... RrU8GT33 Expression boxes, i.e., each element (promoter P) TEFin ,Gene RrU8GT33 and Termination T lip2t The primers and templates used for amplification differ; see Table 2 for details. The IntA site integration plasmid was prepared using a restriction endonuclease from NEB. Pac Following enzyme digestion, the linearized IntA site integration plasmid was recovered by agarose gel electrophoresis. The expression cassette P... TEFin - RrU8GT33 -T lip2t The linearized IntA site integration plasmid was cloned and validated in one step to obtain the positive recombinant plasmid pUC-HUH-IntA-. RrU8GT33。

[0039] Table 2 Primers used for constructing recombinant plasmids from glycosyltransferases of different sources.

[0040] 3. Recombinant plasmid pUC-HUH-IntA- RsUGT72B14 Construction Recombinant plasmid pUC-HUH-IntA- RsUGT72B14 The construction method is the same as pUC-HUH-IntA- RrU8GT33 The difference lies in the fact that RrU8GT33 The expression box was replaced with RsUGT72B14 Expression boxes, i.e., each element (promoter P) TEFin ,Gene RsUGT72B14 and Termination T lip2tThe primers and templates used for amplification are different, as detailed in Table 2.

[0041] 4. Recombinant plasmid pUC-HUH-IntA- AtUGT85A1 Construction Recombinant plasmid pUC-HUH-IntA- AtUGT85A1 Structure as Figure 3 As shown, its construction method is the same as pUC-HUH-IntA- RrU8GT33 The difference is that the expression box is replaced with AtUGT85A1 Expression box, i.e., each element (P) TEFin , AtUGT85A1 and T lip2t The primers and templates used for amplification are different, see Table 2 for details.

[0042] 5. Recombinant plasmid pUC-HUH-IntB- EcTYRA M53I / A354V Construction Recombinant plasmid pUC-HUH-IntB- EcTYRA M53I / A354V Using the IntB site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of IntB. EcTYRA M53I / A354V Gene expression cassette (P TEFin - EcTYRA M53I / A354V -T lip2t Its construction method is similar to that of the recombinant plasmid pUC-A08- EcAROG S180F Similarly, the difference lies in the skeleton, and the expression box is replaced with... EcTYRA M53I / A354V Expression boxes, i.e., each element (promoter P) TEFin ,Gene EcTYRA M53I / A354V and Termination T lip2t The primers and templates used for amplification differ; see Table 3 for details. The IntB site integration plasmid was implanted using a restriction endonuclease from NEB. Pac Following enzyme digestion, the linearized IntB site integration plasmid was recovered by agarose gel electrophoresis. The expression cassette P... TEFin - EcTYRA M53I / A354V -T lip2t The linearized IntB site integration plasmid was cloned and validated in one step to obtain the positive recombinant plasmid pUC-HUH-IntB-. EcTYRA M53I / A354V .

[0043] Table 3 Recombinant plasmid pUC-HUH-IntB- EcTYRA M53I / A354V Primers used in construction

[0044] 6. Recombinant plasmid pUC-HUH-IntC- Year 10 Construction Recombinant plasmid pUC-HUH-IntC- Year 10 Using the IntC site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntC. Year 10 Gene expression cassette (P TEFin - Year 10 -T cyc1t Its construction method is the same as pUC-A08- EcAROG S180F Similarly, the difference lies in the skeleton, and the expression box is replaced with... Year 10 Expression boxes, i.e., each element (promoter P) TEFin ,Gene Year 10 and Termination T cyc1t The primers and templates used for amplification differ; see Table 4 for details. The IntC site integration plasmid was implanted using a restriction endonuclease from NEB. Pac Following enzyme digestion, the linearized IntC site-integrated plasmid was recovered by agarose gel electrophoresis. The expression cassette P... TEFin - Year 10 -T cyc1t The linearized IntC site integration plasmid was cloned and validated in one step to obtain the positive recombinant plasmid pUC-HUH-IntC-. Year 10.

[0045] Table 4 Primers used for constructing recombinant plasmids from phenylpyruvate decarboxylases from different sources.

[0046] 7. Recombinant plasmid pUC-HUH-IntC- ScARO10 I335E Construction Recombinant plasmid pUC-HUH-IntC- ScARO10 I335E The construction method is the same as pUC-HUH-IntC- Year 10 The difference lies in the fact that Year 10 The expression box was replaced with ScARO10 I335E Expression boxes, i.e., each element (promoter P) TEFin ,Gene ScARO10 I335E and Termination T cyc1t The primers and templates used for amplification are different, as detailed in Table 4.

[0047] 8. Recombinant plasmid pUC-HUH-IntD- ScARO10I335E Construction Recombinant plasmid pUC-HUH-IntD- ScARO10 I335E Using the IntD site integration plasmid as a backbone, [the plasmid] was inserted between the upstream and downstream homologous arms of IntD. ScARO10 I335E Gene expression cassette (P TEFin - ScARO10 I335E -T cyc1t ). with pUC-HUH-IntC- ScARO10 I335E Using the template, the primer pair in number 7 of Table 4 was used for amplification to obtain... ScARO10 I335E Expression box (promoter P) TEFin ,Gene ScARO10 I335E and Termination T cyc1t The IntD site-integrating plasmid was processed using a restriction endonuclease from NEB. Pac Following enzyme digestion, the linearized IntD site-integrated plasmid was recovered by agarose gel electrophoresis. The expression cassette P... TEFin - ScARO10 I335E -T cyc1t The linearized IntD site integration plasmid was cloned and validated in one step to obtain the positive recombinant plasmid pUC-HUH-IntD-. ScARO10 I335E .

[0048] 9. Recombinant plasmid pUC-HUH-IntF- ScARO10 I335E Construction Recombinant plasmid pUC-HUH-IntF- ScARO10 I335E Using the IntF site integration plasmid as a backbone, its construction method is the same as pUC-HUH-IntD- ScARO10 I335E The difference lies in the backbone, and the amplification primer pair for the expression cassette is replaced with sequence 8 in Table 4. The IntF site integrator plasmid is then processed using a restriction endonuclease. Pac I. After enzyme digestion, the linearized IntF site integration plasmid was recovered via gel extraction. The expression cassette P... TEFin - ScARO10 I335E -T cyc1t The linearized IntF site integration plasmid was cloned and validated in one step to obtain the positive recombinant plasmid pUC-HUH-IntF-. ScARO10 I335E .

[0049] 10. Recombinant plasmid pUC-HUH-IntE- Year Construction Recombinant plasmid pUC-HUH-IntE- Year Using the IntE site integration plasmid as a backbone, inserts were placed between the upstream and downstream homologous arms of IntE. Year Gene expression cassette (P TEFin -YlTKL-T xpr2t Its construction method is the same as pUC-A08- EcAROG S180F Similarly, the difference lies in the skeleton, and the expression box is replaced with... Year Expression boxes, i.e., each element (promoter P) TEFin ,Gene Year and Termination T xpr2t The primers and templates used for amplification differ; see Table 5 for details. The IntE site integration plasmid was implanted using a restriction endonuclease from NEB. SnB Following enzyme digestion, the linearized IntE site-integrated plasmid was recovered by agarose gel electrophoresis. The expression cassette P... TEFin - Year -T xpr2t The components and linearized IntE site of the plasmid were integrated into the plasmid in a one-step cloning and validation process to obtain the positive recombinant plasmid pUC-HUH-IntE-. YlTKL.

[0050] Table 5 Recombinant plasmid pUC-HUH-IntE- Year Primers used in construction

[0051] 11. Construction of recombinant plasmid pUC-HUH-PPYK-PPYKjd For details on the construction method of the recombinant plasmid pUC-HUH-PPYK-PPYKjd, please refer to Chinese Patent CN120082582A.

[0052] 12. Construction of recombinant plasmid pUC-HUH-PHA2 The recombinant plasmid pUC-HUH-PHA2 uses pUC57-hisG-Ura-hisG as its backbone. EcoR I. Inserted at the restriction site Y. lipolytica In the Po1f Δku70 genome, a 1500bp homologous arm (PHA2-up) upstream of the start codon at the PHA2 site (gene number YALI1_B22549g) is present. KpnA 1500bp homologous arm (PHA2-dm) was inserted downstream of the PHA2 stop codon at the I restriction site. The hisG tag encoding gene and the orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing the promoter P) were also inserted. TEFin orotate nucleoside-5'-phosphate decarboxylase and terminator T xpr2t Between upstream and downstream homologous arms. Y. lipolytica Using Po1f Δku70 genomic DNA as a template, the homologous arm PHA2-up upstream of the start codon at the PHA2 site was amplified using the primers in Table 6. Restriction endonucleases were then used to amplify the amplification. EcoR I. After digesting the plasmid pUC57-hisG-Ura-hisG with enzymes, the linearized pUC57-hisG-Ura-hisG plasmid was recovered by agarose gel electrophoresis. The linearized pUC57-hisG-Ura-hisG plasmid and its upstream homologous arm PHA2-up were cloned in one step to obtain a circular recombinant vector, which was transformed into E. coli DH5α competent cells. The positive recombinant plasmid pUC-HUH-PHA2-up was obtained by screening with ampicillin-resistant plates and verification by colony PCR and sequencing.

[0053] by Y. lipolytica Using Po1f Δku70 genomic DNA as a template, the downstream homologous arm PHA2-dm of the PHA2 stop codon was amplified using the primers in Table 6. Restriction endonucleases were then used to amplify the DNA. Kpn After digesting the plasmid pUC-HUH-PHA2-up with enzymes, the linearized pUC-HUH-PHA2-up plasmid was recovered by agarose gel electrophoresis. The linearized pUC-HUH-PHA2-up plasmid and PHA2-dm were cloned in one step to obtain a circular recombinant vector, which was transformed into *E. coli* DH5α competent cells. The recombinant plasmid pUC-HUH-PHA2 was obtained through ampicillin-resistant plate selection and verified by colony PCR and sequencing. The pUC-HUH-PHA2 plasmid was then digested with the restriction endonuclease NotI from NEB, and the linearized pUC-HUH-PHA2 plasmid was recovered by agarose gel electrophoresis.

[0054] 13. Construction of recombinant plasmid pUC-HUH-TRP3 The recombinant plasmid pUC-HUH-TRP3 uses pUC57-hisG-Ura-hisG as its backbone. EcoR I. Inserted at the restriction site Y. lipolytica In the Po1f Δku70 genome, a 1500bp homologous arm (TRP3-up) upstream of the start codon at the TRP3 site (gene number YALI1_D13963g) is present. KpnA 1500bp homologous arm (TRP3-dm) downstream of the TRP3 stop codon was inserted at the I restriction site. The hisG tag encoding gene and the orotate nucleoside-5'-phosphate decarboxylase encoding gene expression cassette (containing the promoter P) were also included. TEFin orotate nucleoside-5'-phosphate decarboxylase and terminator T xpr2t The upstream and downstream homologous arms are located between each other. The construction method is the same as that of the recombinant plasmid pUC-HUH-PHA2, except that the primers for amplifying the upstream and downstream homologous arms are TPR2-Up-F / R and TPR2-Dm-F / R as shown in Table 6.

[0055] Table 6 Primer sequences used for constructing the knockout plasmid

[0056] Example 2 Construction of recombinant Yersinia lipolyticis (I) Construction (expression) of recombinant bacteria 1-3 RrU8GT33, RsUGT72B14 or AtUGT85A1 ) The recombinant plasmid pUC-HUH-IntA- RrU8GT33 Import Y. lipolytica In Po1f Δku70, RrU8GT33 The expression cassette was integrated into the IntA site of the genome, resulting in recombinant strain 1. It was then subjected to 5-fluoroorotic acid selection pressure, during which a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The pUC-HUH-IntA- RsUGT72B14 Import Y. lipolytica In Po1f Δku70, RsUGT72B14 The expression cassette was integrated into the IntA site of the genome, resulting in recombinant strain 2. It was then subjected to 5-fluoroorotic acid selection pressure, during which a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The pUC-HUH-IntA- AtUGT85A1 Import Y. lipolytica In Po1f Δku70, AtUGT85A1 The expression cassette was integrated into the IntA site of the genome to obtain recombinant bacteria 3. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection.

[0057] The specific method for constructing recombinant bacteria 1 is as follows: Y. lipolytica Po1f Δku70 was cultured overnight in YPD liquid medium, and competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II. The linearized recombinant plasmid pUC-HUH-IntA- was then applied. RrU8GT33 Conversion Y. lipolyticaHomologous recombination was performed in Po1f Δku70 competent cells. Positive clones were selected using SD-Ura selection medium and then identified by PCR. The PCR-identified positive clones were plated on YPD plates containing 5-fluoroorotic acid (5-fluoroorotic acid). Single colonies were then streaked simultaneously on both YPD and SD-Ura plates. Single colonies that grew on the YPD plate but not on the SD-Ura plate were selected, indicating successful loss of the Ura selection marker, resulting in recombinant strain 1.

[0058] The construction methods for recombinant bacteria 2-3 are the same as those for recombinant bacteria 1, except that the recombinant plasmids need to be modified accordingly.

[0059] (II) Construction (expression) of recombinant strain 4 EcAROG S180F ) The recombinant plasmid pUC-A08- EcAROG S180F Imported into recombinant bacteria 3, EcAROG S180F The expression cassette was integrated into the A08 site of the genome, yielding recombinant bacteria 4. The specific method for constructing recombinant bacteria 4 is as follows: Recombinant bacteria 3 were cultured overnight in YPD liquid medium. Competent cells were prepared using the Zymogen Frozen EZ Yeast Transformation Kit II from Zymo Research Corporation, and the linearized recombinant plasmid pUC-A08- was then applied. EcAROG S180F The cells were transformed into competent cells of recombinant bacteria 3 and homologous recombination was performed. Positive clones were selected using SD-Leu screening medium and then identified by PCR. The strains that were correctly identified by PCR were recombinant bacteria 4.

[0060] (III) Construction (expression) of recombinant strain 5 EcTYRA M53I / A354V ) The recombinant plasmid pUC-HUH-IntB- EcTYRA M53I / A354V Imported into recombinant bacteria 4, which had lost Ura. EcTYRA M53I / A354V The expression cassette was integrated into the IntB site of the genome to obtain recombinant strain 5. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The construction of recombinant strain 5 is the same as that of recombinant strain 1, except that the plasmid used for homologous recombination and the host for transformation need to be modified accordingly.

[0061] (iv) Construction (expression) of recombinant bacteria 6-7 Year 10 or ScARO10I335E ) The recombinant plasmid pUC-HUH-IntC- Year 10 Imported into recombinant bacteria 5 that had lost Ura. Year 10 The expression cassette was integrated into the IntC site of the genome, resulting in recombinant strain 6. It was then subjected to 5-fluoroorotic acid selection pressure, during which a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The pUC-HUH-IntC- ScARO10 I335E Imported into recombinant bacteria 5 that had lost Ura. ScARO10 I335E The expression cassette was integrated into the IntC site of the genome to obtain recombinant strain 7. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The construction methods for recombinant strains 6 and 7 are the same as for recombinant strain 1, except that the plasmid used for homologous recombination and the host used for transformation need to be modified accordingly.

[0062] (V) Construction of recombinant bacteria 8-9 (multiple copy expression) ScARO10 I335E ) The recombinant plasmid pUC-HUH-IntD- ScARO10 I335E Imported into recombinant bacteria 7 that had lost Ura. ScARO10 I335E The expression cassette was integrated into the IntD site of the genome, resulting in recombinant strain 8. It was then subjected to 5-fluoroorotic acid selection pressure, during which a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The pUC-HUH-IntF- ScARO10 I335E Imported into recombinant bacteria 8 that had lost Ura. ScARO10 I335E The expression cassette was integrated into the IntF site of the genome to obtain recombinant strain 9. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The construction methods for recombinant strains 8-9 are the same as for recombinant strain 1, except that the plasmid used for homologous recombination and the host used for transformation need to be modified accordingly.

[0063] (vi) Construction (overexpression) of recombinant strain 10 Year ) pUC-HUH-IntE-YlTKL was introduced into recombinant bacteria 8, which had lost Ura. YearThe expression cassette was integrated into the IntE site of the genome to obtain recombinant strain 10. Then, under 5-fluoroorotic acid selection pressure, a HisG tag and a Ura selection marker were lost for use in the next round of Ura selection. The construction method of recombinant strain 10 is the same as that of recombinant strain 1, except that the plasmid used for homologous recombination and the host used for transformation need to be modified accordingly.

[0064] (vii) Construction of recombinant strain 11 (with truncated PYK promoter) The recombinant plasmid pUC-HUH-PPYK-PPYKjd was introduced into recombinant bacteria 10 that had lost Ura. The pyruvate kinase encoding gene... YlPYK The endogenous promoter P PYK P truncated to a length of 300 bp PYK-jd Recombinant strain 11 was obtained.

[0065] The construction method of recombinant strain 11 is the same as that of recombinant strain 1, except that the plasmid used for homologous recombination and the host used for transformation need to be changed accordingly.

[0066] (viii) Construction of recombinant bacteria 12-13 (knockout) PHA2 and TRP3 ) The recombinant plasmid pUC-HUH- PHA2 Importing recombinant bacteria 11, which has lost its Ura component, into the culture medium... PHA2 Gene knockout yielded recombinant bacteria 12, which were then subjected to 5-fluoroorotic acid selection pressure to lose a HisG tag and an Ura selection marker for use in the next round of Ura selection. The recombinant plasmid pUC-HUH- TRP3 Importing recombinant bacteria 12, which has lost its Ura component, into the culture medium... TRP3 Gene knockout yielded recombinant bacteria 13. The construction methods for recombinant bacteria 12-13 are the same as for recombinant bacteria 1, except that the plasmid used for homologous recombination and the host used for transformation need to be modified accordingly.

[0067] Example 3 (a) Cultivation of engineered bacteria and extraction of products The initial strain was Yersinia lipophila (Yersinia lipophila). Y. lipolyticaThe recombinant strain 1-13 in Example 2 (Po1f Δku70) produced rhodioloside. The specific method is as follows: The strain was activated and cultured in YPD liquid medium at 30 ℃ and 220 rpm for 16 h to obtain a seed culture. The seed culture was inoculated into 50 mL of fermentation medium at a 1% v / v inoculation rate and cultured at 30 ℃ and 220 rpm for 5 days to obtain the fermentation broth. The broth was centrifuged at 12000 rpm for 5 min, and the supernatant was used for liquid chromatography to determine the concentration of rhodioloside. The remaining sample was then dried in an oven (75 ℃) to constant weight for biomass measurement. The fermentation medium formulation was: 20 g / L peptone, 10 g / L yeast extract, and 60 g / L glucose, with water as the solvent.

[0068] (II) Qualitative and quantitative analysis of rhodioloside Rhodioloside detection: UV detector, equipped with a C18 reverse-phase column, mobile phase A: 5% (v / v) formic acid, mobile phase B: 100% acetonitrile, mobile phase concentration gradient: 0–15 min, 85%–60% solution A; 15–20 min, 60%–3% solution A; 20–25 min, 3%–85% solution A. Detection wavelength: 280 nm, column oven temperature: 30 ℃, mobile phase flow rate: 0.3 mL / min.

[0069] After 5 days of fermentation, Yarrowia lipolytica Rhodioloside was not detected in Po1f Δku70. The concentrations of rhodioloside in recombinant bacteria 1~13 (YL-1~YL-13) were as follows: Figure 4 As shown, recombinant strain 13 had the highest rhodioloside yield, reaching 1636.8 mg / L, meaning it produced 1636.8 mg of rhodioloside per liter of fermentation broth. Recombinant strain 13 was named *Yarrowia lipolytica* (…). Yarrowia lipolytica XJ-SAL strain.

[0070] Table 7. Rhodioloside production of initial and recombinant strains

[0071] Recombinant strain 13 was inoculated into 50 mL of seed culture (YPD medium) and cultured at 30 °C and 220 rpm for 24 h. The seed culture was then transferred to a 5 L fermenter at an inoculation rate of 5% v / v. 2.5 L of fermentation medium was added to the 5 L fermenter. Fermentation was carried out for 0-48 h, with dissolved oxygen controlled at 20%; for fermentation longer than 48 h, dissolved oxygen was controlled at 0-5%. The pH was maintained constant at 5.5 throughout fermentation until completion. Feed was added twice, at 40 h and 96 h, each time with 800 mL of a mixed aqueous solution containing 11.1 g ammonium sulfate and 900 g glucose per liter. The fermentation temperature was 28 °C, and the culture time was 6 days. The fermentation medium consisted of 60 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone. After 6 days of fermentation, the highest rhodioloside yield of recombinant strain 13 reached 21528.9 mg / L, which is 21528.9 mg of rhodioloside per liter of fermentation broth (Table 7), significantly higher than that of the initial strain and recombinant strain 3.

[0072] This invention provides a method for constructing and applying a recombinant lipophilic yeast that produces high levels of rhodioloside. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing a recombinant lipophilic yeast that produces high levels of rhodioloside, characterized in that, The recombinant Yersinia lipolyticis expressed a glycosyltransferase gene. UGT phenylpyruvate decarboxylase gene ARO10 transketonease gene TKL DAHP synthase mutant gene AroG S180F and bifunctional branched acid mutase / prephenyl acid dehydrogenase mutant gene TyrA M53I / A354V ; At the same time, the prebenzoic acid dehydrase gene was knocked out. PHA2 and anthranilate synthase gene TRP3 ; wherein, the aforementioned UGT Derived from Arabidopsis thaliana Arabidopsis thaliana .

2. The construction method according to claim 1, characterized in that, The aforementioned UGT The nucleotide sequence is shown in SEQ ID NO.

3.

3. The construction method according to claim 1, characterized in that, The aforementioned ARO10 Derived from brewer's yeast Saccharomyces cerevisiae Or Yeast extract Yarrowia lipolytica ; The aforementioned TKL Derived from Yersinia lipolytica Yarrowia lipolytica The aforementioned AroG S180F and TyrA M53I / A354V All were derived from Escherichia coli Escherichia coli.

4. The construction method according to claim 3, characterized in that, Derived from brewer's yeast Saccharomyces cerevisiae of ARO10 The nucleotide sequence is shown in SEQ ID NO.6; Derived from Yersinia lipolytica Yarrowia lipolytica of ARO10 The gene number is YALI1_D08884g; Derived from Yersinia lipolytica Yarrowia lipolytica of TKL The gene number is YALI1_E07744g; Derived from Escherichia coli Escherichia coli of AroG S180F and TyrA M53I / A354V The nucleotide sequences are shown in SEQ ID NO.4~5 respectively.

5. The construction method according to claim 1, characterized in that, The aforementioned PHA2 The gene number is YALI1_B22549g; the aforementioned TRP3 The gene number is YALI1_D13963g.

6. The construction method according to claim 1, characterized in that, The recombinant Yersinia lipophila expressed 1-3 copies of [the substance]. ARO10.

7. The construction method according to claim 1, characterized in that, The recombinant Yersinia lipase gene simultaneously modifies the natural promoter P of the pyruvate kinase gene. PYK Truncation, the truncated P PYK The nucleotide sequence is shown in SEQ ID NO.

7.

8. The recombinant Yersinia lipophila constructed by the construction method according to any one of claims 1 to 7.

9. The application of the recombinant Yersinia lipolytica according to claim 8 in the fermentation production of rhodioloside.

10. The application according to claim 9, characterized in that, The recombinant Yersinia lipolyticis was inoculated into a fermentation medium and fermented at 28-32 °C for 4-7 days to obtain rhodioloside.

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