Genetically engineered bacteria with high yield of elenolic acid and construction method and application thereof

Abstract

This invention discloses a genetically engineered bacterium producing high levels of oleoflavones, its construction method, and its applications, belonging to the fields of genetic engineering and microbial technology. The genetically engineered bacterium for high oleoflavone production is a Streptomyces strain overexpressing the ORF8022 gene (Seq ID No. 1) and / or the ORF8003 gene (Seq ID No. 2). Its construction method includes the following steps: constructing an overexpression plasmid with a strong promoter element driving the expression of the target gene; and transferring the constructed plasmid to a Streptomyces host producing oleoflavones via conjugation transfer. This invention, through genetic engineering, overexpresses the endogenous genes ORF8022 and ORF8003, which are potentially related to oleoflavone biosynthesis, thereby significantly increasing the yield of oleoflavones.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and microbial technology, specifically relating to a genetically engineered bacterium that produces high levels of oleuropein, its construction method, and its application. Background Technology

[0002] Oleoflavone is a class of sixteen-membered ring macrolide antibiotics with a symmetrical structure. It was first isolated from *Streptomyces melanosporus*, and there are currently reports of many different *Streptomyces* strains producing oleoflavone. The absolute configuration of oleoflavone was determined by X-ray crystallography in 1982, and its total chemical synthesis was completed in 1986.

[0003] Studies have shown that oleuropein and its homologues possess excellent biological activity and potential pharmaceutical value: 1) Anti-Gram-positive bacteria activity, such as Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis, Mycobacterium, Enterococcus faecalis, and Enterococcus faecium, although the bactericidal mechanism is not yet clear. It also shows good activity against drug-resistant Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis (MRSA and MRSE), and vancomycin-resistant Enterococcus faecalis and Enterococcus faecium (VRE). It has no activity against Gram-negative bacteria. 2) It has no activity against yeasts and filamentous fungi, but experimental data show that when used in combination, it can significantly enhance the antifungal activity of rapamycin. 3) Anthelmintic activity. Some natural components have anti-malarial activity against Plasmodium falciparum. Some semi-synthetic compounds based on the natural structure of oleuropein compounds, after chemical modification, show strong anthelmintic activity against Caenorhabditis elegans at a concentration of 100 ppm. 4) Anticancer activity. Oleoflavones exhibit moderate cytotoxic activity, inhibiting autophagy, inducing apoptosis, and resisting angiogenesis. 5) Immunosuppressive activity. Several oleoflavone compounds demonstrate immunosuppressive activity, with one component already in preclinical studies for psoriasis, ischemia-reperfusion, and allergies. 6) Some components can inhibit α-glucosidase, exhibiting antiviral activity.

[0004] Based on the chemical structure of oleuropein, it can be inferred that its carbon skeleton is catalyzed by type I polyketide synthase. In 2004, the biosynthetic gene cluster of oleuropein was cloned from *S. malaysiensis* DSM4137 by Professor Leadlay's research group in the UK. This cluster, exceeding 60 kb in length, contains five polyketide synthase genes arranged in a cluster, responsible for the synthesis of the oleuropein macrolide backbone. Seven genes related to glycosyl synthesis and transport are distributed upstream and downstream of the polyketide synthase genes, encoding proteins responsible for the glycosylation of oleuropein. The gene cluster also contains some regulatory and transport genes. In 2015, Professor Taifo's research group in the US further deduced the biosynthetic pathway of oleuropein and the mechanism of formation of nine different components in *Streptomyces* sp. ICBB 9297. The proteins encoded by the five core type I PKS genes contain eight modules, responsible for the synthesis of two linear polyketide chains. In the same year, Professor Leadlay's research group, led by Zhou Yongjun, conducted in-depth research on the gene ela1*, which encodes the independent type II thioesterase domain TE, proving that it is responsible for the cyclization of two chains to form the oleuropein backbone structure. In 2017, Professor Lu Tao and Professor Wen Mengliang's research group at Yunnan University discovered that in S. autolyticus CGMCC0516, a regulatory gene gdmRIII, contained in the gledycin biosynthesis gene cluster, positively regulates gledycin biosynthesis while negatively regulating oleuropein biosynthesis.

[0005] Streptomyces 219807 is the producer of oleuropein, and its yield is generally maintained at around 1 g / L during laboratory shake-flask fermentation. To achieve large-scale preparation and obtain sufficient quantities of pure product for later research, it is necessary to further improve the potency of the producing strain. Summary of the Invention

[0006] The purpose of this invention is to provide a genetically engineered bacterium that produces high levels of oleuropein, its construction method, and its application for the preparation of oleuropein-like compounds.

[0007] The first objective of this invention is to provide a genetically engineered bacterium that produces high levels of oleuropein. This genetically engineered bacterium is obtained by overexpressing at least one of the following genes in a Streptomyces host that produces oleuropein:

[0008] (a) The ORF8022 gene is an NAD(P)-dependent oxidoreductase gene with an undetermined function, and its nucleotide sequence is shown in Seq ID No.1.

[0009] (b) The ORF8003 gene is a transcription factor gene with an undetermined function, and its nucleotide sequence is shown in Seq ID No. 2.

[0010] In one embodiment, the Streptomyces that produces oleuropein is Streptomyces 219807 with accession number CCTCC NO: M2015276. Streptomyces 219807 has been disclosed in Chinese Patent CN104876984A, "A strain producing high-yield oleuropein compounds and its preparation method and application", and was deposited at the China Center for Type Culture Collection on May 4, 2015.

[0011] In one embodiment, the ORF8022 and / or ORF8003 genes are overexpressed in a Streptomyces host using a site-specific integrative vector containing a strong promoter. The site-specific integrative vector is preferably the pSET152 or pIB139 plasmid. The strong promoter is preferably the constitutive promoter hrdBp, whose nucleotide sequence is shown in Seq ID No. 3.

[0012] A second objective of this invention is to provide a method for constructing a genetically engineered bacterium that produces high levels of oleuropein. The method comprises the following steps: constructing an overexpression plasmid that drives the expression of a target gene (ORF8022 gene and / or ORF8003 gene) using a strong promoter element; and transferring the constructed plasmid into a Streptomyces host that produces oleuropein via conjugation transfer to obtain a genetically engineered bacterium that produces high levels of oleuropein. The overexpression plasmid is preferably pSET152 or pIB139 plasmid as a vector. The strong promoter is preferably the constitutive promoter hrdBp.

[0013] Furthermore, the construction of the overexpression plasmid includes any one of the following two methods:

[0014] 1) Construction of the ORF8022 gene overexpression plasmid: The specific steps include: designing primers LXY36F and LXY36R inside the ORF8022 gene in Streptomyces 219807, adding an NdeI restriction site at the start codon ATG, PCR amplifying the ORF8022 gene in Streptomyces 219807 that does not contain the original promoter, digesting the amplified 966bp ORF8022 gene without the promoter with NdeI and PmeI, inserting it into the NdeI and EcoRV sites of the vector pWHU1288 containing the constitutive strong promoter hrdBp, and obtaining plasmid pLXY48.

[0015] The sequences of the primers LXY36F and LXY36R are as follows:

[0016] LXY36F:5'-GGAATTC CATATG GGCCACATCCGAGATCG-3';

[0017] LXY36R: 5'-GTGGCC GTTTAAAC G ACTAGT TCAGTGTCCTTCGGTACGGG-3'.

[0018] 2) Construction of the ORF8003 gene overexpression plasmid: The specific steps include: designing primers LXY40F and LXY40R inside the ORF8003 gene in Streptomyces 219807, adding an NdeI restriction site at the start codon ATG, PCR amplifying the ORF8003 gene in Streptomyces 219807 that does not contain the original promoter, digesting the amplified 2877bp ORF8003 gene without the promoter with NdeI and PmeI, and inserting it into the NdeI and EcoRV sites of the vector pWHU1288 containing the constitutive strong promoter hrdBp to obtain plasmid pLXY51.

[0019] The sequences of the primers LXY40F and LXY40R are as follows:

[0020] LXY40F: 5'-GGAATTC CATATG GTGTTTTCATCGGCCAG-3';

[0021] LXY40R: 5'-GTGGCC GTTTAAAC G ACTAGT TCAGGCGATTTCGTCCACCT-3'.

[0022] The construction of the vector pWHU1288 containing the constitutive strong promoter hrdBp includes the following steps: primers hrdB-pF and hrdB-pR are designed inside the hrdB gene in *Streptomyces cerevisiae* M145. An NdeI restriction site is added at the start codon ATG. The promoter of hrdB in *Streptomyces cerevisiae* M145 is amplified by PCR, as shown in Seq ID No. 3. The amplified 451bp DNA fragment containing hrdBp is digested with XbaI and BamHI and inserted into the XbaI and BamHI sites of the integrative vector pSET152 (commercial vector, GenBank sequence number: AJ414670.1) to obtain plasmid pWHU1288.

[0023] The primer sequences hrdB-pF and hrdB-pR are as follows:

[0024] hrdB-pF: 5'-AATT TCTAGA CCGCCTTCCGCCGGAACG-3';

[0025] hrdB-pR: 5'-AATT GGATCC CATATG CAACCTCTCGGAACGTTG-3'.

[0026] Transplantation into the Streptomyces host refers to: transforming the constructed overexpression plasmid into *E. coli* ET12567 / pUZ8002, and then transferring the plasmid into *Streptomyces* 219807 via biparental conjugation to obtain conjugation transferants. Specific steps include: transforming the constructed overexpression plasmid into *E. coli* ET12567 / pUZ8002; selecting transformants and culturing them overnight in liquid LB medium, followed by inoculation into fresh liquid LB medium containing a final concentration of 30 μg / mL apromycin, 50 μg / mL kanamycin, and 25 μg / mL chloramphenicol; and culturing to OD... 600 The concentration should be 0.4-0.6. Centrifuge 1 mL, discard the supernatant, and collect the bacterial cells. Wash the cells twice with fresh, antibiotic-free LB medium. Resuspend the cells in 500 μL of fresh, antibiotic-free liquid LB medium. Wash the *Streptomyces* 219807 spores twice with 0.05 M pH 8.0 TES solution. Resuspend the spores in 500 μL of pre-germination medium, heat-shock at 50°C for 10 min, and incubate at 37°C in a shaker for 0.5-1 hour. Mix the prepared *E. coli* suspension with the pre-germinated spore suspension and spread it onto SFM agar plates. Incubate at 28°C for 12-17 hours, then cover with 1 mL of sterile water containing apopramycin (final concentration 30 μg / mL) and nalidixic acid (final concentration 25 μg / mL). Incubate at 28°C for 3 days to obtain conjugation transferons.

[0027] A third objective of this invention is to provide the application of the genetically engineered bacteria in the preparation of olive leaf extract.

[0028] A method for preparing olive leaf extract includes the following steps: inoculating the seed culture of the genetically engineered bacteria onto a fermentation medium for fermentation.

[0029] Advantages and beneficial effects of this invention: This invention uses genetic engineering to overexpress the endogenous genes ORF8022 and ORF8003, which are likely related to the biosynthesis of oleuropein. The resulting genetically engineered bacteria showed a 108% and 97% increase in oleuropein yield, respectively, compared to the control strain. This invention provides a new approach for the industrial production of oleuropein. Attached Figure Description

[0030] Figure 1 Schematic diagram of a recombinant plasmid containing the strong promoter hrdBp to overexpress the target gene.

[0031] Figure 2 Recombinant plasmid digestion verification agarose gel electrophoresis image; lane M is the DNA molecular weight standard; lane 1 is pLXY48 digested with NdeI and SpeI; lane 2 is pLXY51 digested with NdeI and SpeI.

[0032] Figure 3 : PCR verification of Streptomyces conjugation transferons; Lane M is the DNA molecular weight standard; Lane 1 is the negative control, i.e., the result of amplification using 219807 total DNA as template and primers hrdB-pF and hrdB-pR; Lane 2 is the product of amplification using 219807::pLXY48 total DNA as template and primers hrdB-pF and hrdB-pR; Lane 3 is the product of amplification using 219807::pLXY51 total DNA as template and primers hrdB-pF and hrdB-pR.

[0033] Figure 4 HPLC detection of fermentation products of wild-type strain 219807; among them, oleuropein was retained at 15.199 minutes.

[0034] Figure 5 Comparison of relative yields of oleuropein from genetically engineered strains. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] The molecular cloning tool Escherichia coli DH5a and the biparental conjugation transfer tool Escherichia coli ET12567 / pUZ8002 are commercial products, and their competent cells were prepared according to *Molecular Cloning: A Laboratory Manual*. The oleuropein-producing strain *Streptomyces 219807*, with accession number CCTCC NO: M2015276, has been disclosed in Chinese patent CN104876984A, "A strain producing high-yield oleuropein compounds and its preparation method and application."

[0038] The primers used in the experiments of this invention are shown in Table 1 below. The primers were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0039] Table 1 Primers used in the experiments of this invention

[0040]

[0041]

[0042] The culture media and reagents used in the experiments of this invention are as follows:

[0043] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, add distilled water to 1000mL, sterilize at 115℃ for 30min.

[0044] LA medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, add distilled water to 1000mL, sterilize at 115℃ for 30min.

[0045] SFM medium: 20g soybean meal powder, 20g mannitol, 20g agar, add distilled water to 1000mL, sterilize at 115℃ for 30min.

[0046] TSBY medium: sucrose 103g, Oxoid tryptophan soup powder 30g, yeast extract 5g, add distilled water to 1000mL, sterilize at 115℃ for 30min.

[0047] Fermentation medium: 10g glucose, 25g dextrin, 20g oat flakes, 10g cottonseed meal, 5g fish meal, 2g yeast extract, 3g CaCO3, add distilled water to 1000mL, sterilize at 115℃ for 30min.

[0048] Spore pre-germination medium: 1% yeast extract, 1% casein amino acids, 0.01M CaCl2.

[0049] TES solution: Weigh 2.292g of tris(hydroxymethyl)aminoethanesulfonic acid (TES) powder, add deionized water to 200mL, adjust pH to 8.0, and sterilize at 121℃ for 20min.

[0050] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0051] Example 1: Construction of a Streptomyces site-specific integrative vector containing the constitutive strong promoter hrdBp

[0052] Primers hrdB-pF and hrdB-pR were designed based on the upstream DNA sequence of the hrdB gene in the genome information of *Streptomyces cerevisiae* M145. Primer hrdB-pF introduced an XbaI restriction site, primer hrdB-pR introduced a BamHI restriction site, and an NdeI restriction site was added at the start codon ATG. This amplified a 451 bp 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 commercially available integrative vector pSET152 to obtain plasmid pWHU1288. DNA sequencing was performed by Wuhan Qingke Biotechnology Co., Ltd. The bacterial strain and plasmid DNA were preserved for later use.

[0053] Example 2: Construction of recombinant plasmid for target gene overexpression

[0054] The genome of Streptomyces 219807, a bacterium that produces oleuropein, was scanned. Analysis of the genome information suggests that the region of genes ORF8000 to ORF8027 on the chromosome may contain the gene cluster for the biosynthesis of oleuropein.

[0055] Total DNA from *Streptomyces 219807*, a producer of oleuropein, was used as a template for PCR amplification using primers LXY33F and LXY33R. A 1886 bp DNA fragment was obtained, containing 1847 bp regions of genes ORF8016 and downstream ORF8017, as shown in Seq ID No. 4, encoding possible glycosyltransferases and epiisomerases, respectively. The PCR was performed using 2× Hieff Canacetylene gels from Shanghai Yisheng Biotechnology Co., Ltd. TM PCR Master Mix high-fidelity enzyme premix system. A 20 μL PCR system includes: template, 1 μL; primer F, 1 μL; primer R, 1 μL; DMSO, 1 μL; sterile deionized water, 6 μL; 2× Hieff Canacetate... TMPCR Master Mix, 10 μL. PCR reaction program: 98℃, 3 min; 98℃, 10 s, 60℃, 20 s, 72℃, 30 s / kb, 30 cycles; 72℃, 5 min. The DNA fragments obtained from PCR amplification were isolated and purified by agarose gel electrophoresis and an Omega Bio-Tek gel extraction kit. The fragments were digested with NdeI and PmeI enzymes for further purification. The vector pWHU1288 constructed in Example 1 was digested with NdeI and EcoRV enzymes for purification. The exogenous fragment and the vector fragment were ligated using T4 ligase. The ligation system was transformed into *E. coli* DH5α competent cells according to *Molecular Cloning: A Laboratory Manual*. The procedure is briefly described as follows: Add 5-10 μL of enzyme ligation system to *E. coli* DH5α competent cells, mix gently, and incubate on ice for 30 minutes; heat shock at 42°C for 90 seconds; incubate on ice for 3 minutes; add 1 mL of LB medium; incubate on a shaker at 37°C for 45 minutes; centrifuge at 6000 rpm for 3 minutes; discard the supernatant, add 300 μL of LB medium to resuspend the precipitate; plate on LA plates containing apopramine at a final concentration of 50 μg / mL, air dry; incubate overnight at 37°C. Single colonies are picked and transferred to LB medium containing apopramine at a final concentration of 50 μg / mL, and incubated overnight on a shaker at 37°C. Plasmids are extracted using an Omega Bio-Tek plasmid extraction kit. After enzyme digestion verification, DNA sequencing by Wuhan Qingke Biotechnology Co., Ltd. confirms consistency with the original sequence. The correct plasmid is named pLXY44, an overexpression plasmid for genes ORF8016 and ORF8017. The bacterial strain and plasmid DNA are preserved for later use.

[0056] Using total DNA from *Streptomyces 219807*, a bacterium that produces oleuropein, overexpression plasmids ORF8018-8019, ORF8022, ORF8023, ORF8024, ORF8003, and ORF8010 were constructed using the same method (see schematic diagram of plasmids). Figure 1 (As shown), and save the correct plasmid and corresponding bacterial strain for later use. The construction process is briefly described below:

[0057] PCR amplification using primers LXY35F and LXY35R yielded a 1706 bp DNA fragment containing a 1675 bp region of the gene ORF8018 and its downstream gene ORF8019, as shown in Seq ID No. 5, encoding two possible ABC transport proteins. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. After confirming correct enzyme digestion, DNA sequencing verified consistency with the original sequence. The correct plasmid was named pLXY47, representing the overexpression plasmid of genes ORF8018 and ORF8019.

[0058] PCR amplification using primers LXY36F and LXY36R yielded a 997 bp DNA fragment containing the 966 bp gene ORF8022, as shown in Seq ID No. 1, encoding a possible NAD(P)-dependent oxidoreductase. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. Digestion of the plasmid with NdeI and SpeI should produce two fragments, 6130 bp and 969 bp respectively. Agarose gel electrophoresis analysis is shown below. Figure 2 As shown in lane 1, the result is consistent with the theoretical value. DNA sequencing verification confirmed that the sequence is identical to the original sequence. The correct plasmid was named pLXY48, which is the overexpression plasmid for the gene ORF8022.

[0059] PCR amplification using primers LXY37F and LXY37R yielded a 1018 bp DNA fragment containing the 987 bp gene ORF8023, as shown in Seq ID No. 6, encoding a possible ketoreductase. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. After confirming correct enzyme digestion, DNA sequencing verified consistency with the original sequence. The correct plasmid was named pLXY50, representing the overexpression plasmid of gene ORF8023.

[0060] PCR amplification using primers LXY38F and LXY38R yielded a 1438 bp DNA fragment containing the 1407 bp gene ORF8024, as shown in Seq ID No. 7, encoding a possible dehydrogenase. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. After confirming correct enzyme digestion, DNA sequencing verified consistency with the original sequence. The correct plasmid was named pLXY49, an overexpression plasmid of gene ORF8024.

[0061] PCR amplification using primers LXY40F and LXY40R yielded a 2908 bp DNA fragment containing the 2877 bp gene ORF8003, as shown in Seq ID No. 2, encoding a potential transcriptional regulatory protein. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. Digestion of the plasmid with NdeI and SpeI should produce two fragments, 6130 bp and 2880 bp respectively. Agarose gel electrophoresis analysis is shown below. Figure 2 As shown in lane 2, the result is consistent with the theoretical value. DNA sequencing verification confirmed that the sequence is identical to the original sequence. The correct plasmid was named pLXY51, which is the overexpression plasmid for gene ORF8003.

[0062] PCR amplification using primers LXY45F and LXY45R yielded a 4978 bp DNA fragment containing the 4947 bp gene ORF8010, as shown in Seq ID No. 8, encoding a polyketide synthase. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. After confirming correct enzyme digestion, DNA sequencing verified consistency with the original sequence. The correct plasmid was named pDQ139, an overexpression plasmid of gene ORF8010.

[0063] In addition, using *Streptomyces cerevisiae* M145 as a template, a 1229 bp DNA fragment containing the 1197 bp gene adpA, encoding a functionally defined global transcriptional regulator, was amplified using primers adpA-F and adpA-R. The relevant sequence is shown in the NCBI database, GenBank accession number 1098226. After isolation and purification, the fragment was digested with NdeI and PmeI, ligated to the vector pWHU1288 (digested and purified with NdeI and EcoRV), and transformed into *E. coli* DH5α. Transformants were cultured, and plasmids were extracted. After confirming correct enzyme digestion, DNA sequencing was performed, confirming consistency with the original sequence. The correct plasmid was named pLXY55, the overexpression plasmid of the adpA gene.

[0064] Example 3: Construction of a high-yield genetically engineered strain of olive leaf extract

[0065] 1. Co-transfer of recombinant plasmids overexpressing the target gene from *Escherichia coli* to *Streptomyces* genus 219807.

[0066] Referring to the *Streptomyces Experimental Operation Manual*, the plasmids constructed in Example 2 were transferred into *Streptomyces* 219807 via biparental conjugation transfer. Experimental data confirmed that the plasmid pWHU2449, which overexpresses the genes sfp and svp from Professor Xudong Qu's research group at Shanghai Jiao Tong University, has the ability to enhance the production of secondary metabolites in *Streptomyces*. The construction process can be found in the reference Benyin Zhang, Wenya Tian, ​​Shuwen Wang, Xiaoli Yan, Xinying Jia, Gregory K. Pierens, Wenqing Chen, Hongmin Ma, Zixin Deng, Xudong Qu, Activation of Natural Products Biosynthetic Pathways via a Protein Modification Level Regulation, ACS Chem. Biol. 2017, 12, 7, 1732–1736. Therefore, the plasmid pWHU2449, which overexpresses the genes sfp and svp, was transferred into *Streptomyces* 219807 via biparental conjugation transfer. A brief procedure is as follows:

[0067] The plasmid from Example 2 was transformed into Escherichia coli ET12567 / pUZ8002 competent cells using the same method as the Escherichia coli DH5α competent cells described above, except that apopramycin, kanamycin, and chloramphenicol were used, with final concentrations of 30 μg / mL, 50 μg / mL, and 25 μg / mL, respectively.

[0068] Escherichia coli ET12567 / pUZ8002 containing the target plasmid was inoculated into 5 mL of LB medium containing apopramycin 30 μg / mL, kanamycin 50 μg / mL, and chloramphenicol 25 μg / mL, and cultured overnight at 37°C with shaking. The overnight cultured E. coli was then transferred at a 1:10 ratio to 5 mL of fresh LB medium containing apopramycin 30 μg / mL, chloramphenicol 25 μg / mL, and kanamycin 50 μg / mL, and cultured at 37°C with shaking until OD. 600 The concentration should be 0.4-0.6. Centrifuge 1 mL, discard the supernatant, and collect the bacterial cells. Wash the bacterial cells twice with fresh LB medium without antibiotics, and suspend the bacterial cells in 500 μL of liquid LB medium for later use.

[0069] Spores of Streptomyces 219807 were suspended in 1 mL of 0.05 M pH 8.0 TES solution, shaken to mix, and centrifuged at 10,000 rpm for 1 min. The supernatant was discarded. This step was repeated once. The spores were resuspended in 500 μL of fresh spore pre-germination medium, heat-shocked at 50 °C for 10 min, and cultured on a shaker at 37 °C for 1 hour for later use.

[0070] The prepared E. coli suspension and the pre-germinated spore suspension were mixed and spread onto SFM agar plates. After incubation at 28°C for 17 hours, the plates were covered with 1 mL of sterile water containing apopramycin (final concentration 30 μg / mL) and nalidixic acid (final concentration 25 μg / mL). After incubation at 28°C for 3 days, the presence of conjugation transferons was observed.

[0071] 2. Total DNA extraction from Streptomyces conjugation transferons

[0072] Pick a single colony of conjugates appearing on the conjugation transfer plate from step 1 and inoculate it onto SFM medium containing apopramycin (50 μg / mL). Incubate at 28°C for 5-7 days, then transfer to TSBY liquid medium containing apopramycin (25 μg / mL) and incubate at 28°C with a shaker for 48 hours. Collect the bacterial cells and extract total DNA. The method for extracting chromosomal DNA is as follows, referring to the *Streptomyces Laboratory Operation Manual*: Place 500 μL of mycelium in a sterilized 1.5 mL centrifuge tube and centrifuge at 10,000 rpm for 2 min, discarding the supernatant; wash the bacterial cells twice with sterile water, centrifuge again, discarding the supernatant; add 500 μL of lysozyme (2 mg / mL), incubate at 37°C for 30 min, inverting to mix every 2 min; add 4% SDS. 25 μL, incubate at 55℃ for 5-10 min until transparent; add 300 μL of phenol-chloroform, vortex to mix, centrifuge at 12000 rpm for 5 min; take 700 μL of the supernatant, add 70 μL of sodium acetate (3M), mix well, then add 770 μL of isopropanol, gently invert 5-6 times, place at -40℃ for 20 min; centrifuge at 12000 rpm for 5 min, discard the supernatant, add 1 mL of 75% ethanol, let stand at room temperature for 1 min, centrifuge at 12000 rpm for 1 min, discard the supernatant, repeat this step once; dry in an oven at 55℃, dissolve in 40-60 μL of sterile ultrapure water, and store at -40℃ for later use.

[0073] 3. PCR verification of Streptomyces conjugation transferons

[0074] The total DNA obtained in step 2 was diluted 20-50 times. Using the diluted solution as a template, the genetically engineered bacteria were verified by PCR using the 2×Taq Master Mix (Dye Plus) kit from Nanjing Novizan Biotechnology Co., Ltd. The 20μL PCR system included: template, 1μL; primer hrdB-pF, 1μL; primer hrdB-pR, 1μL; sterile deionized water, 7μL; 2×Taq Master Mix, 10μL. The PCR reaction program was: 95℃, 3min; 95℃, 15s, 60℃, 15s, 72℃, 30s, 30 cycles; 72℃, 5min. The PCR products were analyzed by agarose gel electrophoresis, and the results are shown below. Figure 3 As shown.

[0075] Using wild-type strain 219807 chromosomal DNA as a negative control, and primers hrdB-pF and hrdB-pR, PCR verification was performed on the total DNA sample. Theoretically, no amplification products were found. The agarose gel electrophoresis results are as follows: Figure 3As shown in lane 1, this is consistent with the theory. PCR verification of the conjugation transferon was performed; correct strains should all show a 451 bp amplification product, and the agarose gel electrophoresis results are consistent with the theoretical value. The agarose gel electrophoresis results for Streptomyces strains 219807::pLXY48 and 219807::pLXY51 are shown below. Figure 3 Lanes 2 and 3 are shown. The correct strains should be preserved for future use.

[0076] Example 4: Fermentation of genetically engineered strains and detection of fermentation products

[0077] 1. Seed Culture: The correct genetically engineered bacterial strain obtained in step 3 of Example 3 was activated on an SFM plate containing apopramycin (50 μg / mL). An appropriate amount of spores was collected and inoculated into 5 mL of TSBY liquid medium containing apopramycin (25 μg / mL). The culture was carried out at 28°C and 220 rpm for 3 days to obtain the seed culture. The wild-type strain (WT) Streptomyces 219807 was used as a control.

[0078] 2. Shake flask fermentation: Inoculate the seed liquid obtained in step 1 into a 250mL shake flask containing 50mL of fermentation medium at an inoculation rate of 10%, and ferment at 28℃ and 220rpm for 8 days.

[0079] 3. Fermentation broth treatment: After centrifugation of the fermentation broth, collect the supernatant. Add an equal volume of ethyl acetate and extract in a shaker at 37°C for 30 min. Separate using a separatory funnel and collect the ethyl acetate phase. Repeat the extraction twice using the same method. Combine the ethyl acetate phases obtained from the three extractions, remove water using anhydrous sodium sulfate, filter through filter paper, evaporate the solvent to dryness, dissolve in 1 mL of chromatographic grade acetonitrile, store at low temperature, and filter through a 0.22 μm microporous membrane before detection.

[0080] 4. HPLC Detection: HPLC analysis conditions: Shimadzu SPD-M20A / LC-20AT was used for HPLC analysis. The chromatographic column was a Thermo Scientific C18 reversed-phase column (250×4.6mm, 5μm). 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; injection volume: 10μL. Gradient elution program: After equilibrating the column with 10% mobile phase B for 20 min, injection began. For the first 0-2 min, the mobile phase B ratio was 10%; for the next 2-15 min, the mobile phase B ratio was 10%-100%; for the next 15-25 min, the mobile phase B ratio was 100%; for the next 25-35 min, the mobile phase B ratio was 10%; and for the next 35-45 min, the mobile phase B ratio was 10%. Signal acquisition lasted for a total of 45 min.

[0081] 5. Detection Results: Taking wild-type strain Streptomyces 219807 as an example, the HPLC detection results are illustrated as follows: Figure 4 As shown, according to the standard, the peak with a retention time of 15.119 min is the compound oleuropein. The peaks with the same retention time for each sample were integrated to calculate the change in oleuropein yield in each genetically engineered bacterial strain. This was repeated three times, and the statistical results are shown below. Figure 5 As shown, the average yield of oleuropein in the wild-type control strain 219807 was 984 mg / L, which was taken as 100%. The yields of oleuropein in strain 219807::pLXY48, which overexpresses the ORF8022 gene, and strain 219807::pLXY51, which overexpresses the ORF8003 gene, increased by 108% and 97%, respectively. The yields of other genetically engineered bacterial strains showed no significant change or decreased significantly: strains with no significant change were strain 219807::pDQ139, which overexpressed the ORF8010 gene; strain 21907::pLXY44, which overexpressed the ORF8016 and ORF8017 genes; strain 219807::pLXY50, which overexpressed the ORF8023 gene; strain 219807::pLXY49, which overexpressed the ORF8024 gene; and strain 219807::pLXY47, which overexpressed the ORF8018 and ORF8019 genes. The strains with a significant decrease were strain 219807::pLXY55, which overexpressed the adpA gene; and strain 219807::pWHU2449, which overexpressed the sfp and svp genes.

[0082] The above description illustrates specific embodiments of the present invention. After reading the above description, any person skilled in the art can make various modifications and alterations within the scope of the technology disclosed in this invention, and all such modifications and alterations should be included within the protection scope of this invention. Therefore, the protection scope of this invention should be determined by the scope of the claims.

Claims

1. A genetically engineered bacterium that produces high levels of oleuropein, characterized in that: The genetically engineered bacteria were obtained by overexpressing the ORF8022 gene, with the nucleotide sequence shown in Seq ID No. 1, in a Streptomyces host that produces oleuropein.

2. The genetically engineered bacterium producing high levels of oleuropein according to claim 1, characterized in that: The Streptomyces that produces oleuropein is Streptomyces 219807 with accession number CCTCC NO: M2015276.

3. The genetically engineered bacterium producing high levels of oleuropein according to claim 1, characterized in that: The ORF8022 gene was overexpressed in Streptomyces hosts using a site-specific integrative vector containing a strong promoter.

4. The genetically engineered bacterium producing high levels of oleuropein according to claim 3, characterized in that: The site-specific integration vector is either pSET152 or pIB139 plasmid.

5. The genetically engineered bacterium producing high levels of oleuropein according to claim 3, characterized in that: The strong promoter is a constitutive promoter hrdBp, whose nucleotide sequence is shown in SEQ ID No.

3.

6. The method for constructing a genetically engineered bacterium producing high levels of oleuropein according to any one of claims 1-5, characterized in that: The process includes the following steps: constructing an overexpression plasmid that drives the expression of the target gene with a strong promoter element, and transferring the constructed plasmid into a Streptomyces host that produces oleuropein through conjugation transfer to obtain a genetically engineered bacterium that produces high levels of oleuropein.

7. The use of the genetically engineered bacteria with high oleoresin production according to any one of claims 1-5 in the preparation of oleoresin.

8. A method for preparing olive leaf extract, characterized in that: The method includes the following steps: inoculating the seed culture of the genetically engineered bacteria that produces high levels of oleuropein according to any one of claims 1-5 onto a fermentation medium for fermentation.

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