CN116333950A - Genetically engineered bacterium for high yield of oleanolic acid, construction method and application thereof - Google Patents

Abstract

The invention discloses a genetic engineering bacterium for high-yield oleanolic acid, and a construction method and application thereof, and belongs to the technical fields of genetic engineering and microorganisms. The genetic engineering bacteria for high-yield oleanolic acid of the invention are streptomycetes which over-express ORF8022 gene (Seq ID No. 1) and/or ORF8003 gene (Seq ID No. 2), and the construction method comprises the following steps: an over-expression plasmid with a strong promoter element for driving the expression of a target gene is constructed, and the constructed plasmid is transferred into a streptomycete host for producing the oleanolic acid through a conjugal transfer method. The invention carries out over-expression on the possible endogenous genes ORF8022 gene and ORF8003 gene related to the biosynthesis of the oleanolic acid through a genetic engineering means, so that the yield of the oleanolic acid is greatly improved.

Description

Genetically engineered bacterium for high yield of oleanolic acid, construction method and application thereof

Technical Field

The invention belongs to the technical fields of genetic engineering and microorganisms, and particularly relates to a genetic engineering bacterium for high-yield oleanolic acid, a construction method and application thereof.

Background

Russulin is a class of sixteen-membered ring macrolide antibiotics with symmetrical structure, originally isolated from Streptomyces melanosporus, and many different strains of streptomyces have been reported to produce russulin. The absolute configuration of oleanolic acid was determined by X-ray crystal examination in 1982, and chemical total synthesis was completed in 1986.

Research shows that the oleanolic acid and the homologues thereof have excellent biological activity and potential patent medicine value: 1) Gram-positive bacterial activity such as Bacillus subtilis, bacillus cereus, staphylococcus aureus, staphylococcus epidermidis, mycobacterium, enterococcus faecalis, enterococcus faecium and the like, and the mechanism of sterilization is not clear. Gram positive bacteria resistant to drugs can also exhibit good activity, such as methicillin resistant staphylococcus aureus and staphylococcus epidermidis (MRSA and MRSE), vancomycin resistant enterococci faecalis and enterococcus faecium (VRE). Has no activity against gram-negative bacteria. 2) The antifungal activity of rapamycin is significantly improved by the combined use of yeasts and filamentous fungi, which are not active, but have been shown by experimental data. 3) Insect repellent activity. Some of the natural components have anti-plasmodium falciparum activity. Some semisynthetic compounds, which are based on the natural structure of the oleanolic compounds and have been chemically modified, exhibit very strong repellent activity against caenorhabditis elegans at a concentration of 100 ppm. 4) Anticancer activity. The obtained olive leaf extract has moderate cytotoxicity, and can inhibit autophagy, induce apoptosis, and resist vascular proliferation. 5) Immunosuppressive activity. A variety of oleanolic compounds exhibit immunosuppressive activity, with certain components having entered preclinical studies of psoriasis, ischemia reperfusion and allergy. 6) Some components can inhibit alpha-glucosidase and have antiviral activity.

Based on the chemical structure of oleanolic acid, the carbon skeleton is presumably catalyzed by type I polyketide synthases. In 2004, the cluster of the biosynthesis genes of oleanolic acid was cloned from S.malaysiensis DSM4137 by the group of Leadlay professor task in England, which was over 60kb in length, and contained a clustered array of 5 polyketide synthase genes responsible for the synthesis of the oleanolic acid macrolide backbone. The polyketide synthase gene is provided with 7 genes related to glycosyl synthesis and transport at the upstream and downstream, and the encoded protein is responsible for glycosylation of the oleanolic leaf extract. The gene cluster also comprises a plurality of regulatory genes and transport genes. The biosynthetic pathway of oleanolic acid, as well as the mechanism by which 9 different components form, was once again speculated by the U.S. professor Taifo task group in Streptomyces sp.icbb 9297 in 2015, the 5 core type I PKS gene encodes a protein containing 8 modules responsible for the synthesis of two linear polyketide chains. In the same year, zhou Yongjun et al, of the uk lead professor task group, conducted intensive studies on the gene ela, which encodes an independent type II thioesterase domain TE, demonstrating that it is responsible for cyclization of both chains to form the olivil backbone structure. In 2017, the university of Yunnan Lu Tao professor and the professor Wen Mengliang discovered that one regulatory gene gdmRIII contained in the geldanamycin biosynthesis gene cluster positively regulates geldanamycin biosynthesis and negatively regulates jerusalem artichoke biosynthesis in S.autolyceus CGMCC 0516.

Streptomyces 219807 is a producing strain of oleanolic acid, and its yield is generally maintained at about 1g/L during laboratory shake flask fermentation. In order to perform large-scale preparation, a sufficient amount of pure product is obtained for later research, and the titer of the bacteria producing the pure product needs to be further increased.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium for high-yield oleanolic acid, a construction method and application thereof, which are used for preparing oleanolic acid compounds.

The first object of the present invention is to provide a genetically engineered bacterium which produces high yields of oleanolic acid. The genetically engineered bacterium is obtained by over-expressing at least one of the following genes in a streptomycete host producing the oleanolic acid:

(a) ORF8022 gene, which is a functionally undetermined NAD (P) -dependent oxidoreductase gene, has a nucleotide sequence shown in Seq ID No. 1;

(b) ORF8003 gene refers to a transcription regulator gene whose function is not determined, and the nucleotide sequence is shown in Seq ID No. 2.

In one embodiment, the streptomyces producing oleanolic acid is a compound having a preservation number of cctccc NO: streptomyces 219807 of M2015276 and Streptomyces 219807 are disclosed in Chinese patent CN104876984A, a strain of high-yield oleanolic acid compounds, a preparation method and application of the compounds, and are preserved in China center for type culture collection (China, type culture collection) on 5-month 4 of 2015.

In one embodiment, the ORF8022 gene and/or ORF8003 gene described above is overexpressed in a Streptomyces host in a site-specific integrative vector containing a strong promoter. The site-specific integration vector is preferably a pSET152 or pIB139 plasmid. The strong promoter is preferably a constitutive promoter hrdBP, the nucleotide sequence of which is shown in Seq ID No. 3.

The second object of the present invention is to provide a method for constructing a genetically engineered bacterium for high production of oleanolic acid, comprising the steps of: constructing an over-expression plasmid for driving the expression of a target gene (ORF 8022 gene and/or ORF8003 gene) by a strong promoter element, and transferring the constructed plasmid into a streptomycete host for producing the oleanolic acid by a joint transfer method to obtain the genetically engineered bacterium for producing the oleanolic acid with high yield. The over-expression plasmid preferably uses pSET152 or pIB139 plasmid as a vector. The strong promoter is preferably the constitutive promoter hrdBP.

Further, the construction of the over-expression plasmid comprises any one of the following two modes:

1) The construction of the ORF8022 gene overexpression plasmid comprises the following specific steps: primers LXY36F and LXY36R are designed inside the ORF8022 gene in streptomyces 219807, an NdeI cleavage site is added at the start codon ATG, the ORF8022 gene without the original promoter in streptomyces 219807 is amplified by PCR, the amplified 966bp ORF8022 gene without the promoter is cleaved by NdeI and PmeI, and NdeI and EcoRV sites of a vector pWHO 1288 containing a constitutive strong promoter are inserted to obtain a plasmid pLXY48.

The sequences of the primers LXY36F and LXY36R are as follows:

LXY36F：5’-GGAATTCCATATGGGCCACATCCGAGATCG-3’；

LXY36R：5’-GTGGCCGTTTAAACGACTAGTTCAGTGTCCTTCGGTACGGG-3’。

2) The construction of the ORF8003 gene overexpression plasmid comprises the following specific steps: primers LXY40F and LXY40R are designed inside the ORF8003 gene in streptomyces 219807, an NdeI cleavage site is added at the start codon ATG, the ORF8003 gene of the streptomyces 219807 without the original promoter is amplified by PCR, the amplified 2877bp ORF8003 gene without the promoter is cleaved by NdeI and PmeI, and NdeI and EcoRV sites of a vector pWHO 1288 containing a constitutive strong promoter are inserted to obtain a plasmid pLXY51.

The sequences of the primers LXY40F and LXY40R are as follows:

LXY40F：5’-GGAATTCCATATGGTGTTTTCATCGGCCAG-3’；

LXY40R：5’-GTGGCCGTTTAAACGACTAGTTCAGGCGATTTCGTCCACCT-3’。

the construction of the vector pWHU1288 containing the constitutive strong promoter hrdBP comprises the following specific steps: primers hrdB-pF and hrdB-pR were designed inside the hrdB gene of Streptomyces coelicolor M145, an NdeI cleavage site was added at the initiation codon ATG, the hrdB promoter of Streptomyces coelicolor M145 was amplified by PCR, and the amplified 451bp DNA fragment containing hrdBP was digested with XbaI and BamHI as shown in Seq ID No.3, and inserted into the XbaI and BamHI sites of the integrative vector pSET152 (commercial vector, SEQ ID No.: AJ 414670.1) to obtain plasmid pWHU1288.

The primer hrdB-pF and hrdB-pR sequences are as follows:

hrdB-pF：5’-AATTTCTAGACCGCCTTCCGCCGGAACG-3’；

hrdB-pR：5’-AATTGGATCC CATATGCAACCTCTCGGAACGTTG-3’。

transfer into a Streptomyces host means: transferring the constructed over-expression plasmid into escherichia coli ET12567/pUZ8002, transferring the plasmid into streptomycete 219807 through two parent conjugation transfer to obtain a conjugation transfer, and specifically comprising the following steps: the constructed over-expression plasmid is transferred into escherichia coli ET12567/pUZ8002, and after the transformant is selected and cultured overnight in a liquid LB culture medium, the liquid LB culture medium is transferred with fresh liquid LB culture medium which contains the final concentration of 30 mug/mL of arabicin, 50 mug/mL of kanamycin and 25 mug/mL of chloramphenicol. Culturing to OD ₆₀₀ 0.4-0.6. 1mL of the supernatant was collected by centrifugation, the cells were washed twice with fresh LB medium containing no antibiotic, and the cells were suspended with fresh 500. Mu.L of liquid LB medium containing no antibiotic for use. Streptomyces 219807 spores were washed twice with 0.05M TES solution pH 8.0, suspended in 500. Mu.L of pregermination medium, heat shocked at 50deg.C for 10min, and shake cultured at 37deg.C for 0.5-1 hr. The treated E.coli suspension and the spore suspension after pregermination were mixed and spread on SFM medium plates. After culturing in an incubator at 28℃for 12-17 hours, the incubator was covered with 1mL of sterile water containing apramycin (final concentration: 30. Mu.g/mL) and nalidixic acid (final concentration: 25. Mu.g/mL), and the incubator was cultured at 28℃for 3 days to obtain a conjunctive transfer.

The third object of the invention is to provide the application of the genetically engineered bacterium in the preparation of the oleanolic acid.

A method of preparing oleanolic acid comprising the steps of: inoculating the seed solution of the genetically engineered bacteria to a fermentation medium for fermentation.

The invention has the advantages and beneficial effects that: the invention carries out over-expression on the possible endogenous gene ORF8022 gene and ORF8003 gene related to the biosynthesis of the oleanolic acid through a genetic engineering means, and the yield of the obtained genetic engineering bacteria is respectively improved by 108 percent and 97 percent compared with that of the control strain oleanolic acid. The invention provides a new idea for the industrial production of the oleanolic acid.

Drawings

Fig. 1: schematic of a recombinant plasmid containing the strong promoter hrdBP over-expressing the gene of interest.

Fig. 2: enzyme cutting of recombinant plasmid to verify agarose gel electrophoresis pattern; lane M is a DNA molecular weight standard; lane 1 shows pLXY48 cut with NdeI and SpeI; lane 2 shows pLXY51 digested with NdeI and SpeI.

Fig. 3: PCR verification of streptomycete joint transferor; lane M is a DNA molecular weight standard; lane 1 is the result of amplification of the negative control, 219807 total DNA as template, primers hrdB-pF and hrdB-pR; lane 2 shows 219807 the amplified products of primers hrdB-pF and hrdB-pR using pLXY48 total DNA as template; lane 3 is 219807-the amplified products of primers hrdB-pF and hrdB-pR using pLXY51 total DNA as template.

Fig. 4: HPLC detection of wild-type strain 219807 fermentation product; wherein the retention time 15.199 minutes is oleanolic acid.

Fig. 5: and comparing the relative yield of the gene engineering strain oleanolic acid.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below in connection with specific embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. It should be noted that, based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without making any inventive effort are within the scope of the present invention.

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

Molecular cloning tool E.coli DH5a and two parental ligation transfer tool E.coli ET12567/pUZ8002 are commercial products whose competent cells were prepared according to the guidelines for molecular cloning experiments. Streptomyces europaea 219807 has a preservation number of CCTCC NO: m2015276, which is disclosed in Chinese patent CN104876984A, a strain for producing high-yield oleanolic acid compounds, and a preparation method and application of the compounds.

The primers involved in the experimental process of the present invention are synthesized by Nanjing Jinsri biotechnology Co., ltd, as shown in Table 1 below.

TABLE 1 primers involved in the experiments of the present invention

The culture medium and the reagent involved in the experimental process of the invention are as follows:

LB medium: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

LA medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 15g of agar, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

SFM medium: 20g of soybean cake powder, 20g of mannitol and 20g of agar, adding distilled water to 1000mL,115 ℃ and sterilizing for 30min.

TSBY medium: sucrose 103g, oxoid tryptone soy soup powder 30g, yeast extract 5g, distilled water to 1000mL,115 ℃ and sterilizing for 30min.

Fermentation medium: 10g of glucose, 25g of dextrin, 20g of oatmeal, 10g of cotton seed powder, 5g of fish meal, 2g of yeast extract and CaCO ₃ 3g, distilled water is added to 1000mL,115 ℃ and sterilized for 30min.

Spore pre-germination medium: yeast extract 1%, casein amino acid 1%,0.01M CaCl ₂ 。

TES solution: 2.292g of tris (hydroxymethyl) aminoethane sulfonic acid (TES) powder was weighed, deionized water was added to 200mL, and pH=8.0 was adjusted and sterilized at 121℃for 20min.

The technical scheme of the invention will be further elaborated in the following in connection with specific embodiments.

Example 1: construction of Streptomyces site-specific integration vector containing constitutive strong promoter hrdBP

Primers hrdB-pF and hrdB-pR were designed based on the upstream DNA sequence of hrdB gene in the genomic information of Streptomyces coelicolor M145, the primer hrdB-pF was introduced with XbaI cleavage site, the primer hrdB-pR was introduced with BamHI cleavage site, and an NdeI cleavage site was added at the initiation codon ATG to amplify 451bp of DNA fragment containing the constitutive strong promoter hrdBP as shown in Seq ID No. 3. The amplified DNA fragment was digested with XbaI and BamHI, and inserted into the XbaI and BamHI sites of the commercial integrative vector pSET152 to give plasmid pWHU1288.DNA sequencing was performed by Wohan engine biotechnology Co. The strain and plasmid DNA were preserved for use.

Example 2: construction of recombinant plasmid for overexpression of target Gene

The genome of Streptomyces fargesii 219807 was scanned and analysis of the resulting genomic information speculated that the region of genes ORF8000 to ORF8027 on the chromosome might contain a cluster of biosynthesis genes for russelin.

The total DNA of Streptomyces ambarius 219807 is used as a template, and PCR amplification is carried out by using primers LXY33F and LXY33R to obtain 1886bp DNA fragment, wherein 1847bp region comprising gene ORF8016 and downstream gene ORF8017 is used for respectively encoding possible glycosyltransferase and epi-isomerase as shown in Seq ID No. 4. Wherein PCR was performed using 2X Hieff Canace from Shanghai, assist, santa Clay ^TM PCR Master Mix high-fidelity enzyme premix system. The 20. Mu.L PCR system included: template, 1 μl; primer F, 1. Mu.L; primer R, 1. Mu.L; DMSO,1 μl; sterile deionized water, 6 μl; 2X Hieff Canace ^TM PCR Master Mix, 10. Mu.L. PCR reaction procedure: 98 ℃ for 3min;98 ℃,10s,60 ℃,20s,72 ℃,30s/kb,30 cycles; 72℃for 5min. The DNA fragment obtained by PCR amplification was isolated and purified by agarose gel electrophoresis and a gel recovery kit from Omega Bio-Tek company. Cutting with NdeI and PmeI, and separating and purifying again. The vector pWHU1288 constructed in example 1 was digested with NdeI and EcoRV, and isolated and purified. The exogenous fragment and the vector fragment were ligated using T4 ligase. The ligation system was transformed into E.coli DH 5. Alpha. Competent cells according to the molecular cloning protocol. The following is a brief description: adding 5-10 mu L of enzyme linked system into the competent cells of Escherichia coli DH5 alpha, gently mixing, and ice-bathing for 30 min; heat-shock at 42 ℃ for 90 seconds; ice bath for 3 minutes; 1mL of LB medium is added; shaking culture at 37 ℃ for 45 minutes; 6000rpm centrifugation3 minutes; the supernatant was discarded, and 300. Mu.L of LB medium was added to suspend the pellet; coating on LA flat plates with the final concentration of the apramycin of 50 mug/mL respectively, and air-drying; the culture was carried out in an incubator at 37℃overnight. Single colonies were picked and transferred to LB medium containing arabinomycin at a final concentration of 50. Mu.g/mL, respectively, and shake cultured overnight at 37 ℃. Plasmid extraction kit from Omega Bio-Tek company. And after the enzyme digestion verification is correct, DNA sequencing verification is carried out by the Wohangaceae biotechnology Co., ltd, and the sequence is consistent with the original sequence. The correct plasmid was designated pllxy 44, the over-expression plasmid of genes ORF8016 and ORF 8017. The strain and plasmid DNA were preserved for use.

The total DNA of Streptomyces farreri 219807 is used as a template, and the overexpression plasmids of ORF8018-8019, ORF8022, ORF8023, ORF8024, ORF8003 and ORF8010 (the plasmid schematic diagram is shown in figure 1) are respectively constructed by the same method, and the correct plasmid corresponding strains are preserved for standby. The construction process is briefly described as follows:

PCR amplification was performed using primers LXY35F and LXY35R to obtain a 1706bp DNA fragment containing a region of 1675bp total of gene ORF8018 and downstream gene ORF8019, as shown in Seq ID No.5, encoding two possible ABC transporters respectively. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. And (3) carrying out DNA sequencing verification after enzyme digestion verification is correct, and conforming to the original sequence. The correct plasmid was designated pllxy 47, the over-expression plasmid of genes ORF8018 and ORF 8019.

PCR amplification was performed with primers LXY36F and LXY36R to obtain a 997bp DNA fragment containing 966bp gene ORF8022 encoding a possible NAD (P) -dependent oxidoreductase as shown in Seq ID No. 1. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. The plasmid was digested with NdeI and SpeI to generate a total of 2 fragments of 6130bp and 969bp, which were analyzed by agarose gel electrophoresis as shown in FIG. 2, lane 1, and were in agreement with the theoretical values. DNA sequencing was verified to be identical to the original sequence. The correct plasmid was designated pLXY48, the over-expression plasmid of gene ORF 8022.

PCR amplification was performed with primers LXY37F and LXY37R to obtain a 1018bp DNA fragment containing the 987bp gene ORF8023 encoding a possible ketoreductase as shown in Seq ID No. 6. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. And (3) carrying out DNA sequencing verification after enzyme digestion verification is correct, and conforming to the original sequence. The correct plasmid was designated pLXY50, the over-expression plasmid of gene ORF 8023.

PCR amplification was performed with primers LXY38F and LXY38R, obtaining a 1438bp DNA fragment containing the 1407bp gene ORF8024, as shown in Seq ID No.7, encoding a possible dehydrogenase. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. And (3) carrying out DNA sequencing verification after enzyme digestion verification is correct, and conforming to the original sequence. The correct plasmid was designated pLXY49, the over-expression plasmid of gene ORF 8024.

PCR amplification was performed using primers LXY40F and LXY40R to obtain a 2908bp DNA fragment containing the 2877bp gene ORF8003, shown in Seq ID No.2, encoding a possible transcriptional regulatory protein. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. The plasmid was digested with NdeI and SpeI to generate a total of 2 fragments of 6130bp and 2880bp, which were analyzed by agarose gel electrophoresis as shown in FIG. 2, lane 2, consistent with theory. DNA sequencing was verified to be identical to the original sequence. The correct plasmid was designated pLXY51, the over-expression plasmid of gene ORF 8003.

PCR amplification was performed using primers LXY45F and LXY45R to obtain a 4978bp DNA fragment containing 4947bp gene ORF8010 encoding a polyketide synthase as shown in Seq ID No. 8. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. And (3) carrying out DNA sequencing verification after enzyme digestion verification is correct, and conforming to the original sequence. The correct plasmid was designated pDQ139, the over-expression plasmid of gene ORF 8010.

In addition, using Streptomyces coelicolor M145 as a template, a 1229bp DNA fragment was amplified using primers adpA-F and adpA-R, comprising a 1197bp gene adpA encoding a functionally defined global transcription regulatory factor, related sequences such as the gene sequence number GenBank in the NCBI database: 1098226. After separation and purification, the mixture is digested with NdeI and PmeI, and then is connected with a vector pWHU1288 digested with NdeI and EcoRV and purified, and escherichia coli DH5 alpha is transformed; transferring and culturing the transformant and extracting the plasmid. And (3) carrying out DNA sequencing verification after enzyme digestion verification is correct, and conforming to the original sequence. The correct plasmid was designated pllxy 55, the over-expression plasmid for the gene adpA.

Example 3: construction of high-yield gene engineering strain of oleanolic acid

1. Recombinant plasmid for over-expressing target gene from colibacillus to streptomycete 219807 indirect transfer

The plasmids constructed in example 2 were transferred into Streptomyces 219807 by two-parent conjugation transfer, respectively, as described in the "Streptomyces laboratory Manual". There are experimental data to demonstrate that plasmid pWHU2449, which overexpresses genes sfp and svp from the university of Shanghai traffic professor Qu Xudong task group, has the ability to promote secondary metabolites of Streptomyces, and is constructed as described in references Benylin Zhang, wenya Tian, shuwen Wang, xiaoli Yan, xinying Jia, gregory K.Pierens, wenqing Chen, hongmin Ma, zixin Deng, xudon Qu Activation of Natural Products Biosynthetic Pathways via a Protein Modification Level Regulation, ACS chem.biol 2017, 12,7, 1732-1736. Thus, plasmid pWHU2449 over-expressing gene sfp and gene svp was transferred into Streptomyces 219807 by two parental ligation transfer, respectively. The brief flow is as follows:

the plasmid of example 2 was transferred into E.coli ET12567/pUZ8002 competent cells in the same way as E.coli DH 5. Alpha. Competent cells described above, except that antibiotics were used, which were apramycin, kanamycin and chloramphenicol, at final concentrations of 30. Mu.g/mL, 50. Mu.g/mL and 25. Mu.g/mL, respectively.

Inoculating Escherichia coli ET12567/pUZ8002 containing objective plasmid into5mL of LB medium containing 30 mug/mL of apramycin, 50 mug/mL of kanamycin and 25 mug/mL of chloramphenicol, and shake-culturing overnight at 37 ℃; coli cultured overnight was inoculated in 5mL of fresh LB medium containing 30. Mu.g/mL of arabinomycin, 25. Mu.g/mL of chloramphenicol and 50. Mu.g/mL of kanamycin at a ratio of 1:10, respectively, and shake-cultured at 37℃to OD ₆₀₀ 0.4-0.6. 1mL of the culture medium was centrifuged to collect the supernatant, and the culture medium was washed twice with fresh LB medium containing no antibiotic, and suspended with 500. Mu.L of liquid LB medium for use.

Spores of Streptomyces 219807 were suspended in 1mL of a 0.05M TES solution of pH 8.0, and the supernatant was removed by shaking, stirring, mixing, and centrifuging at 10000rpm for 1 min. This procedure was repeated once. The spores were resuspended in 500. Mu.L of fresh spore pre-germination medium, heat-shocked at 50℃for 10min and shake-cultured at 37℃for 1 hour for further use.

The treated E.coli suspension and the spore suspension after pregermination were mixed and spread on SFM medium plates. After 17 hours of incubation at 28℃the cells were covered with 1mL of sterile water containing apramycin (final concentration 30. Mu.g/mL) and nalidixic acid (final concentration 25. Mu.g/mL), and after 3 days of incubation at 28℃the presence or absence of the conjugal transfer was observed.

2. Streptomyces zygotic transferase total DNA extraction

The single colony of the zygote appearing on the conjugative transfer plate in step 1 is selected and inoculated on SFM medium containing apramycin (50 mug/mL), cultured for 5-7 days at 28 ℃, transferred to TSBY liquid medium containing apramycin (25 mug/mL) and shake-cultured for 48 hours at 28 ℃, and the total DNA extracted from the bacterial cells is collected. The method for extracting the chromosome DNA is described in the manual of Streptomyces experiment, and the brief flow is as follows: placing 500 μl of mycelium into sterilized 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and discarding supernatant; washing the thalli twice by using sterile water, centrifuging and discarding the supernatant; adding 500 μl lysozyme (2 mg/mL), incubating at 37deg.C for 30min, and mixing at intervals of 2 min; adding 4% SDS 25. Mu.L, and drying at 55deg.C for 5-10min to transparent; adding 300 mu L of phenol-chloroform, stirring and mixing uniformly by vortex, and centrifuging at 12000rpm for 5min; taking 700 mu L of upper liquid, adding 70 mu L of sodium acetate (3M), uniformly mixing, adding 770 mu L of isopropanol, slightly reversing the upper liquid for 5-6 times, and standing at-40 ℃ for 20min; centrifuging at 12000rpm for 5min, discarding supernatant, adding 75% ethanol 1mL, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, discarding upper layer, and repeating the steps once; oven drying at 55deg.C, adding 40-60 μl of sterile ultrapure water, dissolving, and storing at-40deg.C.

3. PCR verification of Streptomyces conjugant

And (3) diluting the total DNA obtained in the step (2) by 20-50 times, and carrying out PCR (polymerase chain reaction) verification on the obtained genetically engineered bacteria by using a 2 XTaq Master Mix (Dye Plus) kit of Nanjinouzan biotechnology Co., ltd. The 20. Mu.L PCR system included: template, 1 μl; primer hrdB-pF, 1. Mu.L; primer hrdB-pR,1 μl; sterile deionized water, 7 μl;2 XTaq Master Mix, 10. Mu.L. PCR reaction procedure: 95 ℃ for 3min;95 ℃,15s,60 ℃,15s,72 ℃,30s,30 cycles; 72℃for 5min. The PCR products were analyzed by agarose gel electrophoresis, and the results are shown in FIG. 3.

The wild-type strain 219807 chromosomal DNA was selected as a negative control, and the primers hrdB-pF and hrdB-pR were used for PCR verification of the total DNA sample, and the amplified product was not theoretically generated, and the agarose gel electrophoresis result is shown in lane 1 of FIG. 3, which is consistent with the theory. PCR verification of the to-be-detected conjugant shows that amplification products of 451bp are detected by the correct strain, and the agarose gel electrophoresis result shows that the amplification products are consistent with the theoretical value. The results of agarose gel electrophoresis of Streptomyces strains 219807:pLXY 48 and 219807:pLXY 51 are shown in FIG. 3, lanes 2 and 3. And (5) preserving the correct strain for later use.

Example 4: fermentation of genetically engineered strains and detection of fermentation products

1. Seed culture: the correct genetically engineered strain obtained in step 3 of example 3 was activated on SFM plates containing apramycin (50. Mu.g/mL), and a suitable amount of spore liquid was collected and inoculated into 5mL of TSBY liquid medium containing apramycin (25. Mu.g/mL) and cultured at 28℃and 220rpm for 3 days to obtain seed liquid. Streptomyces 219807 as a control was used as a wild-type strain (WT).

2. Shaking and fermenting: the seed solution obtained in step 1 was inoculated into a 250mL shaking flask containing 50mL of fermentation medium at an inoculum size of 10%, and fermented at 28℃for 8 days at 220 rpm.

3. And (3) fermentation liquid treatment: and centrifuging the fermentation liquor, and taking supernatant. Adding equal volume of ethyl acetate, extracting in a shaking table at 37 ℃ for 30min, separating by a separating funnel, and collecting ethyl acetate phase. The extraction was repeated twice using the same method. The ethyl acetate phases obtained 3 times are combined, dehydrated by anhydrous sodium sulfate, filtered by filter paper, evaporated to dryness, dissolved in 1mL of chromatographic grade acetonitrile, stored at low temperature and filtered by a microporous filter membrane of 0.22 mu m before detection.

Hplc detection: HPLC analysis conditions: HPLC analysis and detection were performed using Shimadzu SPD-M20A/LC-20AT, with a Thermo Scientific C18 reverse phase column (250X 4.6mm,5 μm). Mobile phase a: ultrapure water, mobile phase B: chromatographic grade acetonitrile, flow rate: 1mL/min, detector: DAD detector, detection wavelength: 252nm, full wavelength scan range: 190-800nm, sample injection amount: 10 mu L. Gradient elution procedure: 10% of mobile phase B is balanced for 20min and then sample injection is started, the proportion of the mobile phase B is 10% -100%, the proportion of the mobile phase B is 15-25min, the proportion of the mobile phase B is 100%,25-35min, the proportion of the mobile phase B is 100% -10%,35-45min, the proportion of the mobile phase B is 10%, and the total signal collection is 45min.

5. Detection result: the HPLC detection result is illustrated by the wild-type strain Streptomyces 219807, and the peak with retention time of 15.119min is the compound oleanolic acid according to the standard as shown in FIG. 4. The peaks of the same retention time of each sample were integrated, the change in the production of oleanolic acid in each genetically engineered strain was calculated, and the results of the statistics are shown in fig. 5. The average yield of oleanolic acid in wild-type control strain 219807 was 984mg/L, which was taken as 100%, and the yields of strain 219807:ply48 and strain 219807:ply51 were increased by 108% and 97% over-expressed gene ORF 8022. The yields of other genetically engineered bacteria strains are not obviously changed or are obviously reduced: the strains without obvious change are strain 219807 of overexpressed gene ORF8010, pDQ139, strain 21907 of overexpressed genes ORF8016 and ORF8017, pLXY44, strain 219807 of overexpressed gene ORF8023, pLXY50, strain 219807 of overexpressed gene ORF8024, strain 219807 of overexpressed genes ORF8018 and ORF8019, pLXY47; the strains with significantly reduced expression of the gene adpA were 219807:pLXY 55 and the strains with overexpression of sfp and svp 219807:pWHU 2449.

The foregoing has described embodiments of the present invention. After reading the above description of the present invention, any person skilled in the art may make various changes and modifications within the technical scope of the present invention, and all those changes and modifications are covered in the scope of the present invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims (8)

1. A genetically engineered bacterium for high yield of oleanolic acid is characterized in that: the genetically engineered bacterium is obtained by over-expressing at least one of the following genes in a streptomycete host producing oleanolic acid: the nucleotide sequence is shown as ORF8022 gene of Seq ID No.1, and the nucleotide sequence is shown as ORF8003 gene of Seq ID No. 2.

2. The genetically engineered bacterium for high yield of oleanolic acid of claim 1, wherein: the streptomycete for producing the oleanolic acid is a streptomycete with a preservation number of CCTCC NO: streptomyces 219807 of M2015276.

3. The genetically engineered bacterium for high yield of oleanolic acid of claim 1, wherein: the ORF8022 gene and/or ORF8003 gene is overexpressed in a Streptomyces host in a site-specific integrative vector containing a strong promoter.

4. The genetically engineered bacterium for high yield of oleanolic acid of claim 3, wherein: the site-specific integration vector is preferably a pSET152 or pIB139 plasmid.

5. The genetically engineered bacterium for high yield of oleanolic acid of claim 3, wherein: the strong promoter is a constitutive promoter hrdBP, and the nucleotide sequence of the strong promoter is shown as SEQ ID No. 3.

6. The method for constructing genetically engineered bacteria of high-yield oleanolic acid of any one of claims 1-5, characterized by comprising the steps of: the method comprises the following steps: and constructing an over-expression plasmid for driving target gene expression by a strong promoter element, and transferring the constructed plasmid into a streptomycete host for producing the oleanolic acid by a conjugal transfer method to obtain the genetically engineered bacterium for producing the oleanolic acid at high yield.

7. Use of the genetically engineered bacterium of high-yielding oleanolic acid of any one of claims 1-5 in the preparation of oleanolic acid.

8. A method for preparing oleanolic acid, characterized in that: the method comprises the following steps: inoculating the seed solution of the genetically engineered bacteria of high-yield oleanolic acid of any one of claims 1-5 onto a fermentation medium for fermentation.

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