CN114410495A - Recombinant yeast engineering bacterium for high-yield friedelin - Google Patents

Abstract

The invention relates to a recombinant yeast engineering bacterium for high yield of friedelin, which overexpresses tHMG1, ERG1, ERG9 and ERG 20; or tmgh 1, ERG1, ERG9 and POS 5; furthermore, BTS1, ROX1, YPL064w and YJL062w genes are knocked out, and the friedelin can be efficiently expressed after the tripterygium wilfordii friedelin coding gene is introduced.

Description

Recombinant yeast engineering bacterium for high-yield friedelin

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant yeast engineering bacterium for producing friedelin.

Background

Friedelin is a type of pentacyclic triterpene component of friedelin type, which is present in the exocuticle portion of the cork or stem of various plants. Pharmacological research in recent years shows that the suberone toolHas antioxidant, antiinflammatory, and blood lipid reducing effects, and can inhibit growth and metastasis of leukemia cells (Chang)^[2]et al, 2020), exhibit antidiabetic effects in rats (Sunil)^[8]et al, 2021), inhibits breast cancer MCF-7 cell growth (Subash-Babu)^[7]P, 2017). In addition, friedelin exerts good insect-resistant effect in plant protection (Baskar)^[1]et al.,2014)。

A series of triterpenoid derivatives taking the friedelin as the skeleton are further separated and identified, have great potential in pharmacological activity excavation, and particularly, an active substance tripterine (celastrol) separated from tripterygium wilfordii has obvious weight-losing effect, thereby becoming a new weight-losing medicine with great potential and being concerned. Has pharmacological activities of resisting rheumatoid arthritis, resisting tumor activity, resisting oxidation, protecting neurons, reducing blood sugar and the like, and is one of 5 natural products which are most likely to be developed into medicaments by being listed in the journal of Cell of the international top-level journal in 2007. However, the content of the friendship components in plants is very low, direct extraction from the bark of the plants causes fatal damage to the plants, and the chemical synthesis method has great challenges due to low efficiency, high consumption, environmental pollution and other outstanding problems. The yeast shares a common pathway upstream of the plant triterpene, namely the biosynthesis pathway of 2, 3-oxidosqualene, and can provide a precursor for the synthesis of 2, 3-oxidosqualene, a triterpene. Meanwhile, the compatibility of yeast expression membrane protein and the feasibility of gene modification enable yeast to become the optimal chassis for triterpene synthesis. The method for realizing the high-value natural products by applying synthetic biology and metabolic engineering and a heterologous reconstruction biosynthesis way in a microbial system is the most efficient and environment-friendly strategy at present, and the construction of the recombinant bacteria capable of producing the friedelin with high yield and the production process have important application values.

Yeast can endogenously synthesize 2, 3-oxidosqualene, provide a common precursor for the biosynthesis of most triterpenoids, then cyclize into different triterpene skeletons by squalene cyclase, and finally form a variety of triterpene intermediates and saponin end products under the post-modification effects of cytochrome P450 oxidase, glycosyltransferase, methyltransferase and the like.The cyclization mechanism is the reaction of the highest degree of rearrangement in the current plants, and FRS genes exist in different plants. Hansen^[3]The UBC7 gene is knocked out from a yeast strain AM238(rox1, dos2, yer134c, vba5, ynr063w, ygr259c) to obtain a strain AM254, and the PdFRS gene which is introduced into the big leaf groundsel and takes root in a plasmid form can generate 0.5 mg.L^-1。Zhou^[9]Et al over-expressed ERG9 and tHMG1 genes in plasmid form in yeast BY4741, simultaneously knock out rox1, ypl062w and yjl064w to obtain recombinant strain ZH1, and introduce TwistC 1T in Tripterygium wilfordii^502EAfter the gene is optimized by a culture medium, 37.07 mg.L can be generated^-1Friedelin. The microbial synthesis of friedelin reported to date has low yield, and the loss is easily caused by a plurality of plasmid forms. The introduction of a plurality of transcription units is generally considered to prolong the growth cycle of the yeast, and the invention gradually increases the amount of the friedelin produced by the dominant strains GH3, GD3 and GQ3 after integrating a plurality of transcription units at the chromosome level and knocking out the alternative inhibition gene, but the phenomenon of prolonging the growth cycle does not occur.

Disclosure of Invention

The first aspect of the invention provides a yeast engineering bacterium for high-yield production of friedelin.

The recombinant yeast includes a friedelin synthase encoding gene and overexpresses in the recombinant yeast:

(1) tmgh 1, ERG1, and ERG9 genes; and

(2) any one or more selected from ERG20, POS5 or UPC2.1 gene;

the friedelin synthase is selected from friedelin synthase from Maytenus spinosus, friedelin synthase from Populus tremula, or friedelin synthase from Tripterygium wilfordii.

In a preferred embodiment, the recombinant yeast engineering bacterium with high yield of the friedelin contains a friedelin synthase coding gene and overexpresses:

(1) tmgb 1, ERG1 and ERG 9; and

(2) selected from any one of ERG20, or POS5, or ERG20 and UPC2.1, or POS5 and UPC 2.1;

the friedelin synthase is selected from friedelin synthase from Maytenus spinosus, friedelin synthase from Populus tremula, or friedelin synthase from Tripterygium wilfordii.

In a more preferred embodiment, the recombinant yeast engineering bacterium with high yield of the friedelin of the invention contains a friedelin coding gene and overexpresses:

tmgh 1, ERG1, ERG9 and ERG 20; or tmgh 1, ERG1, ERG9 and POS 5;

in the present invention, the friedelin synthase derived from Maytenus spinosus or the friedelin synthase derived from Populus tremuloides is an enzyme disclosed in the art, and the amino acid sequence can be obtained by searching in GeneBank database, for example, the amino acid sequence of the friedelin synthase derived from Maytenus tremuloides such as GeneBank: APG38073.1, its coding gene GenBank: KX 147270.1; suberone synthase amino acid sequences from aspen such as GeneBank: ART66198.1, the coding gene of which is as in GeneBank: KY 931453.1.

In a preferred embodiment, the friedelin synthase is a friedelin synthase selected from Tripterygium wilfordii (abbreviated as Twoc 1)^T502E) Said Twsc 1^T502EThe amino acid sequence of (a) is as shown in SEQ ID NO: 2, encoding the sequence shown in SEQ ID NO: 2 (or called tripterygium wilfordii friedelin synthase coding gene, abbreviated as TwoC 1)^T502E). In a specific embodiment, the tripterygium wilfordii friedelin synthase encoding gene TwoC 1^T502EHas the sequence shown in SEQ ID NO: 1.

In a specific embodiment of the present invention, genes encoding genes of different sources of friendship synthase, namely, genes encoding GH1 osc1 (amino acid sequence shown in SEQ ID NO: 2) were introduced into 4 initial recombinant bacteria, that is, GH1 (overexpressed tmgb 1, ERG20, ERG1, ERG9 and UPC2.1), GH2 (overexpressed tmg 1, POS5, ERG1, ERG9 and UPC2.1), GH3 (overexpressed tmg 1, ERG20, ERG1 and ERG9), GH4 (overexpressed tmg 1, POS5, ERG1 and ERG9), respectively, and it was found that a friendship synthase (amino acid sequence shown in SEQ ID NO: 2) derived from Tripterygium wilfordii^T502E(the gene sequence is shown as SEQ ID NO: 1), and the wood boltThe ketone yield was significantly higher than that of the friedelin synthase-encoding gene MiFRS (gene sequence shown in GenBank: KX147270.1) derived from Maytenus spinosus (Maytenus ilicifolia) and that of the friedelin synthase-encoding gene PdFRS (gene sequence shown in Genbank: KY931453.1) derived from Populus davidiana. Therefore, in a preferred embodiment of the present invention, the gene encoding friedelin synthase of the recombinant yeast engineering bacterium with high friedelin yield described in the present invention is preferably the gene encoding friesco 1 derived from Tripterygium wilfordii^T502E. Introduction of Twsc 1^T502EThe yield of the friedelin in the initial recombinant strain after gene recombination is GH1>GH2>GH4>GH3。

In the second aspect of the present invention, on the basis of the first aspect of the present invention, there is provided a recombinant engineered yeast strain for producing friedelin with relatively higher efficiency, which is the engineered yeast strain obtained in the first aspect of the present invention, and further knock-out genes of BTS1 and ROX 1.

In a specific embodiment of the invention, four initial recombinant bacteria, namely GH1, GH2, GH3 and GH4, are screened from the first aspect of the invention, BTS1 and ROX1 genes in the strain are further knocked out, strains GD1, GD2, GD3 and GD4 are sequentially obtained, and a friedelin synthase encoding gene TwinC 1 is introduced^T502EAnd testing the expression yield of the friedelin, and finding that the friedelin yield is improved after the genes of BTS1 and ROX1 are knocked out by the initial GH1 strain and GH3 strain, the friedelin yield is inversely reduced after the genes of BTS1 and ROX1 are knocked out by the GH2 strain and the GH4 strain, and the expression of the friedelin can not be detected even in the GD4 strain.

In still another embodiment of the present invention, in the initial recombinant bacteria, namely GH1, GH2, GH3 and GH4, BTS1, ROX1, YPL062w and YJL064w genes in the strain are further knocked out to obtain optimized bacteria GQ1, GQ2, GQ3 and GQ4, and a friedelin synthase encoding gene TOSC 1 is introduced^T502EThe expression yield of the friedelin is tested, and the friedelin yield of strains GQ1 and GQ3 is further improved and is higher than that of GD1 and GD3, and the friedelin synthase encoding gene TwOSC1 is introduced into GQ4^T502EThen even losing activity, the expression of friedelin can not be detected. The yield of the friedelin in the GQ2 strain is not improved, but is inversely reduced.

The yeast engineering bacteria used in the invention can be derived from GEN.PK series saccharomyces cerevisiae or BY series saccharomyces cerevisiae, preferably BY series saccharomyces cerevisiae, such as BY4741 saccharomyces cerevisiae, BY4742 saccharomyces cerevisiae or BY-T20 saccharomyces cerevisiae.

The third aspect of the invention provides a method for constructing the high-yield suberone yeast engineering bacteria.

The construction method of the high-yield friedelin engineering bacteria comprises overexpression at a yeast chromosome level, such as a two-step method; a knockout method comprising a bypass gene, such as CRISPR-Cas9 technology; introduction of the enzyme friedelin synthase, for example in the form of a plasmid with a strong promoter. Integrating a module of 4 transcription units (wherein the module one contains truncated gene tHMG1 coding for 3-hydroxy-3-methylglutaryl coenzyme A reductase and ERG20 coding for FPP synthase, and the module two contains gene ERG9 coding for squalene synthase and gene ERG1 coding for squalene monooxygenase) in a saccharomyces cerevisiae chromosome by a modular two-step assembly method by using a type-S endonuclease, and introducing a friedelin synthase gene TwoC 1^T502EObtaining recombinant strain GH3 with high yield of friedelin; integrating a module of 4 transcription units (wherein the module one contains truncated gene tHMG1 encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase and POS5 gene encoding NADH kinase, and the module two contains ERG9 gene encoding squalene synthase and gene ERG1 encoding squalene monooxygenase) in a saccharomyces cerevisiae chromosome by a modular two-step assembly method using type-S endonuclease, and introducing a friedelin synthase gene TwoC 1^T502EObtaining recombinant strain GH4 with high yield of friedelin; integrating a module III (containing a truncated gene tHMG1 for coding 3-hydroxy-3-methylglutaryl coenzyme A reductase and a transcription regulator UPC2.1 for enabling yeast to absorb exogenous alcohols under aerobic conditions) of 2 transcription units in a recombinant strain GH1 by a modular two-step assembly method by using a type II S endonuclease to obtain a recombinant strain GH3 with high yield of friedelin; by utilizing a type-S endonuclease and a modular two-step assembly method, a module III (containing a truncated gene tHMG1 for coding 3-hydroxy-3-methylglutaryl coenzyme A reductase and a transcription regulator UPC2.1 for enabling yeast to absorb exogenous alcohols under aerobic conditions) with 2 transcription units is integrated in a recombinant strain GH2, so that high yield is obtainedRecombinant strain GH4 of friedelin; through a CRISPR-Cas9 technology, triterpene shunt inhibitory genes BTS1, ROX1, YPL062w and YJL064w are knocked out from recombinant bacteria GH1 to obtain recombinant bacteria GQ 1.

Drawings

FIG. 1 is a schematic diagram of the construction of a gRNA expression cassette.

FIG. 2 is a graph showing the comparison of the expression levels of friedelin obtained by introducing friedelin synthases from different sources into the initial recombinant strain.

FIG. 3 variation of the various genes in Yeast, introduction of the Gene for the enzyme friec 1^T502EThe expression yield of friedelin is plotted in comparison.

FIG. 4 is a graph of the expression yield of friedelin after the optimized recombinant strain GQ1 is cultured by adopting an optimized culture medium.

FIG. 5 is a graph showing the growth of BY4741 and recombinant strains.

Detailed Description

The present invention will be further illustrated by the following specific examples.

The experimental procedures used in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The yeast referred to in each example is Saccharomyces cerevisiae BY4741 in order to enable those skilled in the art to better understand the present invention.

Example 1 integration of multiple endogenous genes into the Yeast genome

The present study used a modular two-step integration (M2S) technique to integrate multiple transcription units on the yeast chromosome. First, integration of overexpression Module at the chromosomal level in Yeast

The sequence truncated gene encoding 3-hydroxy-3-methylglutaryl coenzyme a reductase gene tmg 1, the ERG20 gene encoding FPP synthase, the ERG9 gene encoding squalene synthase, the ERG1 gene encoding squalene monooxygenase and the POS5 gene encoding NADH kinase were downloaded from the yeast genome database (SGD) (yeastgenome. The gene sequence of UPC2.1 was obtained from the literature (Dai)^[4]et al.,2012,Hu^[5]et al, 2020). According to the instruction operation of a yeast genome extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd.), the genome of the Saccharomyces cerevisiae strain BY4741 is extracted as a template, and the Open Reading Frame (ORF) of the overexpressed gene is amplified. Then, based on the sequence information of the genes pTDH3, pADH1, TPI1t, PGit, tHMG1 and ERG20, primers were designed with recognition sites for type IIS restriction enzyme Bsa I (GGTCTCNNNNN). Promoter P_TDH3-P_ADH1A terminator T_TPI1-T_PGIGene tHMG1 and gene ERG20 in the form of plasmids P1 and T1 (plasmids P1 and T1 see Li^[12]et al, 2016) and BY4741 as templates, using

PCR amplification was performed with high fidelity DNA polymerase (New England Biolab, USA). The PCR product was recovered using Gene JET gel extraction kit (Beijing Quanyujin Bio Inc., China). The Golden Gate reaction was performed on the purified DNA fragments with T4 DNA ligase as follows: at 37 deg.C for 3min and 16 deg.C for 4min, performing 25 cycles, then at 50 deg.C for 5min and at 80 deg.C for 5min, and finally maintaining at 4 deg.C. The reaction product was transformed into E.coli Trans-T1 competent cells, cultured at 37 ℃ and the recombinant plasmid was sequenced to correct module I (P) overexpressing tHMG1 and ERG20_TDH3-tHMG1-T_TPI1And P_ADH1-ERG20-T_PGI) The integration of (2). Module II (P) containing tHMG1 and POS5 was constructed in the same manner_TDH3-tHMG1-T_TPI1And P_ADH1-POS5-T_PGI) Module III containing the ERG9 and ERG1 genes (P)_PCK1-ERG9-T_ADH1And P_TEF2-ERG1-T_CYC1) Module IV (P) containing tHMG1 and UPC2.1 genes_FBA-tHMG1-T_FBAAnd P_HXT7-UPC2.1-T_PDC1)。

And constructing head-tail homologous arms required by integration. Primers were designed based on sequence information for L1, L4, pESC-His and Saccharomyces cerevisiae chromosome XVI-15 site (15site 1:15 site-1-F/R; His: His (15site-1) -F/R; L1: L1(15site-1) -F/R; L4: L4-15site 2-F/R; 15site 2:15site 2-F/R). Plasmids T1 and T3 and genomic DNA were used as templates

PCR was performed with high fidelity DNA polymerase (New England Biolab, USA) to clone 15site1, His, L1, L4, and 15site2 gene fragments. The PCR products were recovered and subjected to overlap PCR to construct head-to-tail homology arms.

Finally, these overexpression modules are integrated into the yeast genome in various combinations. The transcription unit module, the selectable marker and the integrated homology arm module were transformed into s.cerevisiae BY4741 cells BY electroporation transfection. Competent cells were prepared as follows: single colonies were inoculated in 4mL liquid YPD medium and centrifuged at 8000g for 1 min to collect the pellet at OD600 ═ 0.6-1.0. The resulting cell pellet was washed twice with 1mL of pre-cooled sterile water and then incubated in 4mL of transformation reagent (10mM LiAc, 10mM DTT, 0.6M sorbitol, 10mM Tris-HCl pH 7.5) for 20 minutes at 25 ℃. The treated cells were harvested by centrifugation and washed twice with 1mL ice-chilled 1M sorbitol buffer and resuspended in 100. mu.L sorbitol buffer. Cells containing 500ng of the DNA fragment were electroporated at 200. omega. at 25. mu.F at 3kV, and incubated for 2 hours at 30 ℃ in 1mL sorbitol buffer, and then plated on selective medium for 2-3 days. After a yeast BY4741 chromosome is transformed BY integrating an overexpression module, a recombinant strain GH3 containing a module I and a module III, a recombinant strain GH4 containing a module II and a module III, a recombinant strain GH1 containing a module I, a module III and a module IV and a recombinant strain GH2 containing a module II, a module III and a module IV are obtained.

Example 2CRISPR/Cas9 technique knockdown of genes on competing pathways

gRNA expression vector p426-SNR52p-gRNA. CAN1.Y-SUP4t (ref. No.43803), Cas9 expression vector p414-TEF1p-Cas9-CYC1t (43802), universal vector pTY-U01 for gene knockout and pTY-U02 are stored in the laboratory (construction method reference Hu)^[5]et al, 2020). Specific gRNAs targeting BTS1, ROX1, YPL062w and YJL064w genes were designed using an open source tool (http:// yeasttricion. tnw. tudelft. nl) and effective target sequences were screened. All gRNA targets used in this studyThe sequences are shown in Table 1. To obtain single gRNAs, equimolar solutions of 24nt oligonucleotides F and R were mixed and annealed to generate a double stranded insert with overhangs at both ends. Single gRNA-expressing plasmid (Engler) was constructed by inserting a double-stranded oligonucleotide fragment into the AarI recognition site of pTY-U01 using the Golden Gate Assembly method^[10]et al.,2014；Lee^[11]et al, 2015). Similar to the construction method of the single gRNA plasmid, BTS1-gRNA was efficiently inserted into the plasmid pTY-U02 to construct a 2gRNA expression plasmid.

TABLE 1oligo gRNA and repair primers

To obtain a strain of a particular genotype, double-stranded oligonucleotide fragments (ds-Oligo) are used to mediate homologous recombination repair. Homologous repair fragments of ROX1, BTS1, YPL062w and YJL064w were amplified from the yeast BY4741 genome (300 bp upstream and downstream of the ORF of the gene) and spliced BY overlap PCR, with primers shown in Table 1. Next, the Cas9 expression vector P414-Leu-TEF1P-Cas9-CYC1t was introduced into four different strains, GH1, GH2, GH3, and GH4, according to the procedures of the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, Calif., USA). According to different knock-out objectives, a total of 500ng of gRNA expression plasmid was mixed with 1 μ g of the corresponding homologous repair fragment, respectively, and electroporated into competent cells containing Cas9 plasmid. The plasmid information for gRNA construction is listed in table 2, and the electroporation conditions were the same as described above. After 2-3 days of culture at 30 ℃ on SD medium without histidine and leucine, four mutant colonies were selected from each plate, genomic DNA was extracted as a PCR template, and the PCR product was verified BY DNA sequencing, with genomic DNA of strain BY4741 as a negative control. The gRNA plasmid carrying the URA3 marker was then removed with 5-fluoroorotic acid and processed according to Mans^[6]And Hu^[5]In an et al study (Mans et al, 2015; Hu et al, 2020)Description of (3) Cas9 plasmid was removed. ROX1 and BTS1 are knocked out from initial recombinant bacteria GH1, GH2, GH3 and GH4 to obtain recombinant bacteria GD1, GD2, GD3 and GD4, and ROX1, BTS1, YPL062w and YJL064w are knocked out to obtain recombinant bacteria GQ1, GQ2, GQ3 and GQ 4.

TABLE 2gRNA plasmid information Table

EXAMPLE 3 construction of a friedelin synthase expression vector

MiFRS (GenBank: KX147270.1) derived from Maytenuusicifolia and PdFRS (Genbank: KY931453.1) derived from Populus davidiana were synthesized by the company Ruibiotech and cloned into the cloning site (BamHI/SalI) of pESC-Leu plasmid. FRSs sequences were downloaded from the National Center for Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov/gorf. html) database. Codon optimized plasmid pYES2-TwOSC1 from T.willfordii^T502EStored in the laboratory (see Zhou for construction method)^[9]et al, 2019). These three recombinant vectors were transformed into modified strains using the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA, USA), and the empty pYES2 vector and pESC-Leu vector were transformed into Yeast as controls. Positive clones were screened on corresponding solid culture plates and confirmed by colony PCR and DNA sequencing.

EXAMPLE 4 fermentation of recombinant strains in shake flasks

Positive clones were cultured in shake flasks (100mL) containing 30mL SC-His-Ura and SC-His-Leu medium containing 2% glucose and incubated at 200rpm and 30 ℃ for 2 days. Then, the cells were collected and induced in 30mL of SC-Ura-His (2% galactose instead of glucose) medium, and further cultured at 200rpm at 30 ℃ for 72 hours. Containing pYES 2-TOSC 1^T502EThe dominant strain is placed in a culture medium containing 5% of glucose, 1% of yeast extract, 3% of peptone and 0.8% of KH₂PO₄And 0.6% MgSO₄·7H₂Culturing in O medium at 30 deg.C and 220rpm for 2 days, collecting cells, and culturing in a medium containing 5% galactose, 1% yeast extract, 3% peptone, and 0.8% KH₂PO₄And 0.6% MgSO₄·7H₂O at 30 ℃ for 72 hours at 200 rpm. Finally, 10mL of yeast cells were collected, boiled with 10mL of 20% KOH and 50% EtOH for 15 minutes, and then the supernatant was extracted with 15mL of n-hexane, sonicated for 30 minutes, and repeated twice. All extracts were combined and the solvent recovered under reduced pressure on a rotary evaporator.

Example 5

Initial recombinant bacteria (GH1, GH2, GH3 and GH4) containing different friendship synthase genes are subjected to fermentation culture according to the fermentation conditions of example 4, the content of the friendship is determined, and the test results are shown in FIG. 2, and the results show that after different friendship synthase genes are inserted, the friendship yield is the highest in GH1, the next is GH2, the next is GH4 and the yield of GH3 is the lowest. The strain contains the coding gene TwoC 1 of the friedelin synthase of thunder god vine^T502EThe yield of the friedelin is obviously higher than that of the friedelin synthase encoding gene MiFRS containing the Maytenus spinosa or the friedelin synthase encoding gene PdFRS containing the populus tremuloides.

Example 6

The strains which are further optimized, namely initial recombinant strains GH1, GH2, GH3 and GH4 are knocked out BY ROX1 and BTS1 to obtain recombinant strains GD1, GD2, GD3 and GD4, or ROX1, BTS1, YPL062w and YJL064w are knocked out to obtain recombinant strains GQ1, GQ2, GQ3 and GQ4, BY4741 and ZH1(ZHou 1)^[9]Etc., ERG9 and tHMG1 genes were overexpressed in BY4741, and rox1, ypl062w and yjl064w were knocked out at the same time) as a control, and a friedelin synthase-encoding gene TwoC 1 was introduced into these bacteria^T502EThe fermentation culture was performed according to the fermentation conditions of example 4, and the yield of friedelin was measured in each strain, and the results are shown in FIG. 3, which indicates that: the yield of the friedelin of the initial recombinant bacteria GH1, GH2, GH3 and GH4 is higher than that of the strain ZH 1. The yield of friedelin in strains of GD1 and GD3 obtained by knocking out genes of BTS1 and ROX1 for initial recombinant bacteria GH1 and GH3 is further improved, while the yield of friedelin is inversely reduced for strains of GD2 and GD4 obtained by knocking out genes of BTS1 and ROX1 for initial recombinant bacteria GH2 and GH4, and GD4 strains are inactivated even after introducing friedelin synthase genes, so that friedelin detection cannot be carried outThe yield of (a); on the basis of strains GD1, GD2, GD3 and GD4, YPL062w and YJL064w genes are further knocked out to obtain strains GQ1, GQ2, GQ3 and GQ4, the yield of friedelin of the strains GQ1 and GQ3 is further improved, particularly the yield of GQ1 is remarkably improved, the activity of the strain GQ4 is lost after the friedelin synthase gene is introduced, the yield of friedelin cannot be detected, and a small amount of friedelin is expressed in GQ 2.

Example 7 production of friedelin by GQ1 bacterium in optimized Medium

The modified recombinant strain GQ1 is cultured in an optimized YPD medium (namely, the YPD medium contains 5% of glucose, 1% of yeast extract, 3% of peptone and 0.8% of KH)₂PO₄,and 0.6％MgSO₄·7H₂O, YPD-optimized Medium reference Zhou^[9]et al, 2019) for 48h, adding 5% galactose to continue the induction culture for 72h, and obtaining the result shown in FIG. 4, wherein the yield of the recombinant strain GQ1 capable of producing friedelin in the YPD optimized culture medium reaches 63.91 mg/L.

Example eight growth curves of recombinant strains

Culturing the original strain BY4741 and all the modified recombinant strains in YPD medium for 48h, adding 5% galactose, and further inducing and culturing. Samples were taken at 12h intervals, and the OD600 was measured by an ultraviolet spectrophotometer to prepare a growth curve. Compared with the original strain BY4741, the recombinant strain shows an advantageous growth state, especially after galactose induction, the OD600 is higher than BY4741, the growth cycle is not prolonged due to genome integration over-expression of genes, the growth cycle is still maintained at 48h after induction and enters a plateau stage, and only the OD600 of GH4 is lower than BY4741 and enters the plateau stage at the initial stage of induction.

Reference to the literature

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Sequence listing

<110> university of capital medical science

<120> recombinant yeast engineering bacterium for high yield of friedelin

<130> TQZX2022-ZL006

<141> 2022-01-05

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2298

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtggaagt tgaaagttgc cgaaagaggt aacgctccat attctgaata cttgtacact 60

accaacgact tctctggtag acaaacttgg gaatttgatc caaatgctgg tactccacaa 120

gaattggcta aagttgaaga agccagaaga aagttcaccg aagatagaca tactgttaag 180

ccagcttcag atttgttgtg gatgatgcaa ttcatgcgtg agaagaactt caagcaaact 240

attccaccag ttaggttggg tgaagaggaa caagttactt acgaagattt gactactgct 300

ttgactagga ctaccaattt ctttaccgct ttacaagctt ctgatggtca ttggccagct 360

gaaaatggtg gtgtttcttt tttcttgcca ccattcatct tcagcttgta cattactggt 420

cacctgaact ctattattac cccagagtac agaaaagaga tcctgagatt catctacaac 480

caccaaaatg aagatggtgg ttggggtatt catatcgaag gtcattctac tatgttcggt 540

actgctttct cttacgtctg cttgagaatt ttgggtatcg aagttgatgg tggtaaggat 600

aatgcttgtg ctagagctag aaagtggatt ttggatcatg gtggtattac ctatatgcca 660

tcttggggta aaacctggtt gtctattttg ggtgtttatg attggtacgg ctgtaatcca 720

atgccacctg aattttggtt gttgccatct tacttgccaa ttcatccagc taagatttgg 780

tgttactgca gaatggttta catgcccatg tcttacttgt acggtaaaag attcgttggt 840

ccaatcactc cattgatctt gcaattgaga gaagaattgc atacccaacc attccacgaa 900

attcaatggc gtcaaactag acatagatgt gctaaagagg acttgtacta cccacactct 960

ttgattcaag atttcatctg ggattccttg tacgttgctt ctgaaccatt attgactaga 1020

tggccattga acaagattag agaaaaggct ttggctaagg ccatggaaca tattcattat 1080

gaggacgaaa actccaggta cattaccatt ggttgtgttg aaaaagcctt gtgtatgttg 1140

tgttgctggg ttgaagatcc aaactctgac tacttcaaga aacacttggc tagaattcca 1200

gattacttgt gggttgccga agatggtatg aaggttcaat cttttggttc ccaattgtgg 1260

gatgctactt ttggttttca agctttggtt gcttctaact tgaccgaaga tgaagttggt 1320

ccagctttgg caaaagctta cgatttcatt aagaagtccc aagtcaagga taacccatct 1380

ggtgattttg aatccatgca taggcatatc tctaaaggtt cttggacctt ctctgatcaa 1440

gatcatggtt ggcaattgtc tgattgcact gctgaagctt tgaagtgttg tttgttggct 1500

gctgaaatgc cacaagaagt tgttggagaa aaaatgaagc ctgaatgggt ttacgaagcc 1560

atcaacatta tcttgtcctt gcaatctaaa tccggtggtt tggctggttg ggaacctgtt 1620

agagctggtg aatggatgga aattttgaac ccaatggaat tcttggagaa catcgttatc 1680

gaacacacct acgttgaatg taccggttct tctattattg ccttcgtgtc tctgaaaaag 1740

ttgtacccag gtcatagaac taaggacatc gataacttca ttagaaacgc catcaggtac 1800

ttggaagatg ttcaatatcc agatggttct tggtatggta actggggtat ttgtttcatc 1860

tactctacca tgtttgcctt aggtggttta gctgctactg gtagaactta cgataattgc 1920

caagctgtta gaagaggtgt cgacttcata ttgaagaacc agtctgatga cggtggatgg 1980

ggtgaatctt atttgtcttg tccaagaaag gtttacaccc cattggatgg tagaagatct 2040

aacgttgttc aaactgcttg ggctatgttg ggtttgttgt atgctggtca agctgaaagg 2100

gatccaactc cattgcatag aggtgcaaaa gttttgatca actaccagat ggaagatggc 2160

ggttacccac aacaagaaat tactggtgtt ttcaagatga actgcatgtt gcattacccc 2220

atctacagaa atgcttttcc aatttgggct ttgggtgaat accgtaaaag agttccattg 2280

ccatctaagg gttactaa 2298

<210> 2

<211> 765

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Trp Lys Leu Lys Val Ala Glu Arg Gly Asn Ala Pro Tyr Ser Glu

1 5 10 15

Tyr Leu Tyr Thr Thr Asn Asp Phe Ser Gly Arg Gln Thr Trp Glu Phe

20 25 30

Asp Pro Asn Ala Gly Thr Pro Gln Glu Leu Ala Lys Val Glu Glu Ala

35 40 45

Arg Arg Lys Phe Thr Glu Asp Arg His Thr Val Lys Pro Ala Ser Asp

50 55 60

Leu Leu Trp Met Met Gln Phe Met Arg Glu Lys Asn Phe Lys Gln Thr

65 70 75 80

Ile Pro Pro Val Arg Leu Gly Glu Glu Glu Gln Val Thr Tyr Glu Asp

85 90 95

Leu Thr Thr Ala Leu Thr Arg Thr Thr Asn Phe Phe Thr Ala Leu Gln

100 105 110

Ala Ser Asp Gly His Trp Pro Ala Glu Asn Gly Gly Val Ser Phe Phe

115 120 125

Leu Pro Pro Phe Ile Phe Ser Leu Tyr Ile Thr Gly His Leu Asn Ser

130 135 140

Ile Ile Thr Pro Glu Tyr Arg Lys Glu Ile Leu Arg Phe Ile Tyr Asn

145 150 155 160

His Gln Asn Glu Asp Gly Gly Trp Gly Ile His Ile Glu Gly His Ser

165 170 175

Thr Met Phe Gly Thr Ala Phe Ser Tyr Val Cys Leu Arg Ile Leu Gly

180 185 190

Ile Glu Val Asp Gly Gly Lys Asp Asn Ala Cys Ala Arg Ala Arg Lys

195 200 205

Trp Ile Leu Asp His Gly Gly Ile Thr Tyr Met Pro Ser Trp Gly Lys

210 215 220

Thr Trp Leu Ser Ile Leu Gly Val Tyr Asp Trp Tyr Gly Cys Asn Pro

225 230 235 240

Met Pro Pro Glu Phe Trp Leu Leu Pro Ser Tyr Leu Pro Ile His Pro

245 250 255

Ala Lys Ile Trp Cys Tyr Cys Arg Met Val Tyr Met Pro Met Ser Tyr

260 265 270

Leu Tyr Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Leu Gln

275 280 285

Leu Arg Glu Glu Leu His Thr Gln Pro Phe His Glu Ile Gln Trp Arg

290 295 300

Gln Thr Arg His Arg Cys Ala Lys Glu Asp Leu Tyr Tyr Pro His Ser

305 310 315 320

Leu Ile Gln Asp Phe Ile Trp Asp Ser Leu Tyr Val Ala Ser Glu Pro

325 330 335

Leu Leu Thr Arg Trp Pro Leu Asn Lys Ile Arg Glu Lys Ala Leu Ala

340 345 350

Lys Ala Met Glu His Ile His Tyr Glu Asp Glu Asn Ser Arg Tyr Ile

355 360 365

Thr Ile Gly Cys Val Glu Lys Ala Leu Cys Met Leu Cys Cys Trp Val

370 375 380

Glu Asp Pro Asn Ser Asp Tyr Phe Lys Lys His Leu Ala Arg Ile Pro

385 390 395 400

Asp Tyr Leu Trp Val Ala Glu Asp Gly Met Lys Val Gln Ser Phe Gly

405 410 415

Ser Gln Leu Trp Asp Ala Thr Phe Gly Phe Gln Ala Leu Val Ala Ser

420 425 430

Asn Leu Thr Glu Asp Glu Val Gly Pro Ala Leu Ala Lys Ala Tyr Asp

435 440 445

Phe Ile Lys Lys Ser Gln Val Lys Asp Asn Pro Ser Gly Asp Phe Glu

450 455 460

Ser Met His Arg His Ile Ser Lys Gly Ser Trp Thr Phe Ser Asp Gln

465 470 475 480

Asp His Gly Trp Gln Leu Ser Asp Cys Thr Ala Glu Ala Leu Lys Cys

485 490 495

Cys Leu Leu Ala Ala Glu Met Pro Gln Glu Val Val Gly Glu Lys Met

500 505 510

Lys Pro Glu Trp Val Tyr Glu Ala Ile Asn Ile Ile Leu Ser Leu Gln

515 520 525

Ser Lys Ser Gly Gly Leu Ala Gly Trp Glu Pro Val Arg Ala Gly Glu

530 535 540

Trp Met Glu Ile Leu Asn Pro Met Glu Phe Leu Glu Asn Ile Val Ile

545 550 555 560

Glu His Thr Tyr Val Glu Cys Thr Gly Ser Ser Ile Ile Ala Phe Val

565 570 575

Ser Leu Lys Lys Leu Tyr Pro Gly His Arg Thr Lys Asp Ile Asp Asn

580 585 590

Phe Ile Arg Asn Ala Ile Arg Tyr Leu Glu Asp Val Gln Tyr Pro Asp

595 600 605

Gly Ser Trp Tyr Gly Asn Trp Gly Ile Cys Phe Ile Tyr Ser Thr Met

610 615 620

Phe Ala Leu Gly Gly Leu Ala Ala Thr Gly Arg Thr Tyr Asp Asn Cys

625 630 635 640

Gln Ala Val Arg Arg Gly Val Asp Phe Ile Leu Lys Asn Gln Ser Asp

645 650 655

Asp Gly Gly Trp Gly Glu Ser Tyr Leu Ser Cys Pro Arg Lys Val Tyr

660 665 670

Thr Pro Leu Asp Gly Arg Arg Ser Asn Val Val Gln Thr Ala Trp Ala

675 680 685

Met Leu Gly Leu Leu Tyr Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro

690 695 700

Leu His Arg Gly Ala Lys Val Leu Ile Asn Tyr Gln Met Glu Asp Gly

705 710 715 720

Gly Tyr Pro Gln Gln Glu Ile Thr Gly Val Phe Lys Met Asn Cys Met

725 730 735

Leu His Tyr Pro Ile Tyr Arg Asn Ala Phe Pro Ile Trp Ala Leu Gly

740 745 750

Glu Tyr Arg Lys Arg Val Pro Leu Pro Ser Lys Gly Tyr

755 760 765

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gatctaacaa ttcgtcatgt aaat 24

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aaacatttac atgacgaatt gtta 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gatccacttg tagtatatct aact 24

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaacagttag atatactaca agtg 24

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatctcgctt taactttacc gttt 24

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cattattcca gaaaatacta atact 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttagttaaag ggaatatagt ataat 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gacaagccaa tactgaaaga aagaa 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcggaaaaat taggtcatat actcc 25

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tcttcgtcgt cgtcttcgtc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtagggctaa cgactcagcg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgtactcaca actttccgcg 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ggtggcagaa gagacctcac 20

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gggaaacgcc tggtatcttt a 21

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tgtgctgcaa ggcgattaag 20

Claims (10)

1. A recombinant yeast with high yield of friedelin comprises a friedelin synthase coding gene, wherein the recombinant yeast comprises:

(1) over-expression of tmgh 1, ERG1 and ERG 9; and

(2) over-expression of any one or more selected from ERG20, POS5, or UPC 2.1;

the friedelin synthase is selected from friedelin synthase from Maytenus spinosus, friedelin synthase from Populus tremula, or friedelin synthase from Tripterygium wilfordii.

2. The high-yield cork ketone recombinant yeast according to claim 1, comprising a cork ketone synthase encoding gene, wherein said recombinant yeast:

(1) over-expression of tmgh 1, ERG1 and ERG 9; and

(2) over-expression is selected from any one group of ERG20, or POS5, or ERG20 and UPC2.1, or POS5 and UPC 2.15;

the friedelin synthase is selected from friedelin synthase from Maytenus spinosus, friedelin synthase from Populus tremula, or friedelin synthase from Tripterygium wilfordii.

3. The high-yield cork ketone recombinant yeast according to claim 2, comprising a cork ketone synthase encoding gene, wherein said recombinant yeast:

over-expression of tmg 1, ERG1, ERG9 and ERG 20; or overexpresses tmg 1, ERG1, ERG9 and POS 5;

the friedelin synthase is a friedelin synthase derived from Tripterygium wilfordii.

4. The recombinant yeast for high yield of friedelin according to claim 3, wherein the friedelin synthase from Tripterygium wilfordii has the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

5. The high-yield cork ketone recombinant yeast according to claim 4, wherein the coding gene of the cork ketone synthase of Tripterygium wilfordii has the amino acid sequence of SEQ ID NO: 1.

6. The recombinant engineered saccharomyces cerevisiae producing high yield of friedelin according to any one of claims 1-5, wherein the genes BTS1 and ROX1 are knocked out.

7. The recombinant yeast engineering bacterium for high yield of friedelin according to claim 6, which knock out YPL064w and YJL062w genes.

8. The recombinant yeast engineering bacterium for high-yield friedelin according to any one of claims 1 to 7, wherein the yeast is a BY-series saccharomyces cerevisiae, such as BY 4741.

9. A method of producing friedelin, the method comprising:

transferring the recombinant yeast engineering bacteria for producing the friedelin into a culture medium, fermenting, extracting, separating and fermenting a bacterial liquid to obtain a target product friedelin, wherein the recombinant yeast engineering bacteria comprises a friedelin synthase coding gene and has the following characteristics:

(1) over-expression of tmg 1, ERG1, ERG9 and ERG 20; or overexpresses tmg 1, ERG1, ERG9 and POS 5; and the number of the first and second groups,

(2) the BTS1, ROX1, YPL064w and YJL062w genes are knocked out;

the friedelin synthase is derived from tripterygium wilfordii and has an amino acid sequence shown in SEQ ID NO2, and the coding gene of the friedelin synthase is shown in SEQ ID NO: 1 is shown.

10. The method according to claim 9, wherein the medium is YPD medium, and induction culture is continued for 72h by adding 5% galactose to YPD medium for 48 h.

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