Application of phosphopantetheinyltransferase B3D0, its encoding gene, recombinant vector containing the gene, and recombinant bacteria

Abstract

The present invention relates to the application of phosphopantetheinyltransferase B3D0, its encoding gene, recombinant vectors containing the gene, and recombinant bacteria, belonging to the field of genetic engineering technology. To increase the yield of natural products such as polyketides and non-ribosomal peptides, the present invention utilizes bioinformatics analysis and other means to mine a highly efficient phosphopantetheinyltransferase B3D0 from a database. The amino acid sequence of the enzyme is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the enzyme is shown in SEQ ID NO.2. By constructing a recombinant vector and recombinant bacteria containing the enzyme encoding gene, the present invention discovered that overexpressing the enzyme can effectively increase the yield of natural products such as polyketides, polyethers, and non-ribosomal peptides.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to applications of phosphopantetheinyltransferase B3D0, a gene encoding the same, a recombinant vector containing the gene, and recombinant bacteria. Background Art

[0002] Polyketides are a large class of natural products with a wide range of sources, a wide variety of types, diverse structures, and rich biological activities. They are an important source for the creation of clinical drugs and green natural product pesticides. Streptomyces is the main industrial producer of polyketide natural product pesticides. However, the metabolic regulatory network of Streptomyces is complex, and several key mechanisms affecting the biosynthesis of natural products such as polyketides have not yet been thoroughly elucidated, resulting in slow development of high-yield strains. The fermentation industry faces common problems such as low product yield and high production costs, which seriously hinders the development and application of polyketide compounds and is an urgent problem to be solved in the field of polyketide natural product research.

[0003] Acyl-carrier protein (ACP) is an essential structural unit for the function of polyketide synthase (PKS), which transfers the chain between enzymes acting on substrates. To function, ACP needs to be inactive in the apo-form ( apo -) into the active full protein form ( Holo -), phosphopantetheinyl transferase (PPTase) is the enzyme that catalyzes this reaction, transferring the phosphopantetheinyl transfer arm (PPant) of coenzyme A to the serine residue of ACP, completing the activation of ACP. Therefore, PPTase is a key enzyme in the function of PKS and is also a key enzyme in the biosynthesis of natural products such as polyketides and non-ribosomal peptides.

[0004] Currently, PPTase, an important catalytic element, has been rarely studied for its high efficiency, which can be used for strain modification. This has resulted in a shortage of catalytic elements and a limited selection of PPTase elements for efficient natural product synthesis. Therefore, the discovery of highly efficient PPTases is of great value for increasing the yield of natural products such as polyketides and non-ribosomal peptides. Summary of the Invention

[0005] To increase the yield of natural products such as polyketides and non-ribosomal peptides, the present invention used bioinformatics analysis and other means to mine a highly efficient PPTase B3D0 (Uniprot protein accession number is A0A2H5B3D0) from the database. By constructing a recombinant vector and recombinant bacteria containing the enzyme encoding gene, it was found that overexpressing the enzyme can effectively increase the yield of natural products such as polyketides, polyethers, and non-ribosomal peptides.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0007] The first object of the present invention is to provide an application of phosphopantetheinyltransferase B3D0 in promoting the synthesis of natural products, wherein the amino acid sequence of the phosphopantetheinyltransferase B3D0 is shown in SEQ ID NO.1, and the natural product is a polyketide compound, a polyether compound or a non-ribosomal peptide compound.

[0008] A second object of the present invention is to provide a use of a gene encoding phosphopantetheinyltransferase B3D0 in promoting the synthesis of natural products, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2; the natural product is a polyketide compound, a polyether compound, or a non-ribosomal peptide compound.

[0009] A third object of the present invention is to provide a recombinant vector containing a gene encoding phosphopantetheinyl transferase B3D0 for use in promoting the synthesis of natural products, wherein the nucleotide sequence of the gene encoding phosphopantetheinyl transferase B3D0 is shown in SEQ ID NO. 2; the natural product is a polyketide compound, a polyether compound, or a non-ribosomal peptide compound.

[0010] In one embodiment of the present invention, the method for constructing the recombinant vector comprises the following steps: constructing a recombinant expression vector pSET28a-B3D0 containing a gene encoding phosphopantetheinyl transferase B3D0, performing double enzyme digestion on the plasmid pSET152-PermE* carrying the constitutive promoter PermE* using two restriction endonucleases, XbaI and EcoRI, to obtain a linear vector backbone LpSET152-1; amplifying the pSET28a-B3D0 vector using the pSET28a-B3D0 vector as a template. B3D0 Gene fragments with homology arms were assembled using the Gibson method. B3D0 The gene fragment was assembled with the linear vector backbone LpSET152-1 to obtain the recombinant vector pSET152-PermE*-B3D0.

[0011] In one embodiment of the present invention, the amplification B3D0 The primers used for the gene fragment are the upstream primer B3D0-F having a nucleotide sequence as shown in SEQ ID NO.4 and the downstream primer B3D0-R having a nucleotide sequence as shown in SEQ ID NO.3.

[0012] The fourth object of the present invention is to provide a use of a recombinant bacterium containing the above-mentioned encoding gene or the above-mentioned recombinant vector in promoting the synthesis of natural products, wherein the natural products are polyketide compounds, polyether compounds or non-ribosomal peptide compounds.

[0013] In one embodiment of the present invention, the method for constructing the recombinant bacteria comprises the following steps: constructing an overexpression integration plasmid encoding the phosphopantetheinyltransferase B3D0 gene, transforming the overexpression integration plasmid into Escherichia coli, and then introducing the overexpression integration plasmid into the starting strain through an inter-genus conjugative transfer experiment to obtain the recombinant bacteria.

[0014] In one embodiment of the present invention, the Escherichia coli is ET12567 (pUZ8002).

[0015] In one embodiment of the present invention, the starting strain is Streptomyces icebergii ( Streptomyces bingchenggensis )BC-101-4, Streptomyces coelicolor ( Streptomyces coelicolor )M145, Streptomyces avermitilis ( Streptomyces avermitilis ) S0, Streptomyces crassa ( Streptomyces rimosus ) M4018, Saccharopolyspora spinosa ( Saccharopolyspora spinosa )NRRL 18395, Streptomyces roseosporus ( Streptomyces roseosporus )NRRL 11379, Streptomyces venezuelae ( Streptomyces venezuelae ) ISP5230 or Streptomyces albus ( Streptomyces albus )IPPDNR.

[0016] In one embodiment of the present invention, when the starting strain is Streptomyces bingchengensis BC-101-4, the recombinant bacteria is used to increase the yield of milbemycin or nanchangmycin; when the starting strain is Streptomyces coelicolor M145, the recombinant bacteria is used to increase the yield of actinomycin; when the starting strain is Streptomyces avermitilis S0, the recombinant bacteria is used to increase the yield of avermectin; when the starting strain is Streptomyces fissures M4018, the recombinant bacteria is used to increase the yield of oxytetracycline; when the starting strain is Saccharopolyspora spinosa NRRL 18395, the recombinant bacteria is used to increase the yield of spinosad; when the starting strain is Streptomyces roseospore NRRL 11379, the recombinant bacteria is used to increase the yield of daptomycin; when the starting strain is Streptomyces venezuelae ISP5230, the recombinant bacteria is used to increase the yield of daunorubicin; when the starting strain is Streptomyces albus IPPDNR, the recombinant bacteria is used to increase the yield of daunorubicin.

[0017] Beneficial effects of the present invention:

[0018] The present invention uses bioinformatics analysis and other means to mine a highly efficient PPTase B3D0 from a database. By constructing a recombinant vector and recombinant bacteria containing the enzyme encoding gene, a total of 8 high-yield recombinant strains carrying the highly efficient PPTase element B3D0 are obtained. The actinomycetin production of the recombinant strain Streptomyces coelicolor M145-B3D0 was increased by 76.47% compared with the starting strain M145, reaching 218.42 mg / L; the milbemycin production of the recombinant strain Streptomyces bingchengensis BC-101-4-B3D0 was increased by 43.41% compared with the starting strain BC-101-4, reaching 1398.63 mg / L, and the nanchangmycin production was increased by 45.33% compared with the starting strain BC-101-4, reaching 1698.29 mg / L; the avermectin production of the recombinant strain Streptomyces avermitilis S0-B3D0 was increased by 28.17% compared with the starting strain S0, reaching 2.73 g / L; the polymyxin production of the recombinant strain Saccharopolyspora spinosa NRRL18395-B3D0 was increased by 30.21% compared with the starting strain NRRL 18395, reaching 35.42 mg / L; the oxytetracycline production of the recombinant strain Streptomyces crisscrossing M4018-B3D0 increased by 69.90% compared with the starting strain M4018, reaching 2.07 g / L; the daptomycin production of the recombinant strain Streptomyces roseosporus NRRL 11379-B3D0 increased by 71.98% compared with the starting strain NRRL11379, reaching 389.43 mg / L; the daptomycin production of the recombinant strain Streptomyces venezuelae ISP5230-B3D0 increased by 39.04% compared with the starting strain ISP5230, reaching 334.99 μg / mL; the daunorubicin production of the recombinant strain Streptomyces albus IPPDNR-B3D0 increased by 27.27% compared with the starting strain IPPDNR, reaching 33.73 μg / mL. The above results indicate that the discovered PPTase element B3D0 can effectively improve the catalytic efficiency of polyketide synthase and non-ribosomal peptide synthetase, thereby increasing the production of type I and II polyketide compounds, polyether compounds and non-ribosomal peptide compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 For genes B3D0 Schematic diagram of the construction of the overexpression integration plasmid pSET152-PermE*-B3D0;

[0020] Figure 2 The figure shows the results of the in vitro catalytic reaction of PPTase B3D0; Figure 2Figure a is the SDS-PAGE run result of the purified substrate ACP, M is Marker, 1 is the elution buffer with a concentration of 250 mM, 2 is the elution buffer with a concentration of 500 mM, Figure 2 Figure b is the SDS-PAGE run result of the purified PPTase B3D0, M is Marker, 1 is the elution buffer with a concentration of 250 mM, 2 is the elution buffer with a concentration of 500 mM, Figure 2 Figure c is the result of HPLC detection of the prosthetic group state of ACP;

[0021] Figure 3 The figure shows the test results of milbemycin production of the recombinant strains BC-101-4-B3D0, BC-101-4-C and the original strain BC-101-4 of Streptomyces bingchengensis;

[0022] Figure 4 The figure shows the test results of avermectin production of the recombinant strains S0-B3D0 and S0-C of Streptomyces avermitilis and the starting strain S0;

[0023] Figure 5 The graph shows the test results of the spinosad production of the recombinant Saccharopolyspora spinosa strains NRRL 18395-B3D0, NRRL 18395-C and the starting strain NRRL18395;

[0024] Figure 6 The figure shows the test results of the actinomycetourin production of the recombinant Streptomyces coelicolor strains M145-B3D0, M145-C and the original strain M145;

[0025] Figure 7 The figure shows the test results of oxytetracycline production of the recombinant strains M4018-B3D0 and M4018-C of Streptomyces crassifolia and the original strain M4018;

[0026] Figure 8 The figure shows the test results of the yield of degamycin of the recombinant strains ISP5230-B3D0, ISP5230-C and the original strain ISP5230 of Streptomyces venezuelae;

[0027] Figure 9 The figure shows the test results of daunorubicin production of the recombinant strains IPPDNR-B3D0 and IPPDNR-C of Streptomyces albus and the starting strain IPPDNR;

[0028] Figure 10 The figure shows the test results of the nanchangmycin production of the recombinant strains BC-101-4-B3D0 and BC-101-4-C of Streptomyces bingchengensis and the original strain BC-101-4;

[0029] Figure 11 This is a graph showing the test results of daptomycin production by the recombinant Streptomyces roseosporus strains NRRL 11379-B3D0, NRRL 11379-C and the original strain NRRL11379. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to specific embodiments and the accompanying drawings. It should be noted that the embodiments mentioned below are only applicable to explaining the present invention, but are not intended to limit the scope of the present invention. The embodiments mentioned below are only some embodiments of the present invention and not all embodiments. In this field, if other technicians do not make creative work, the embodiments they obtain are protected by the present invention.

[0031] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, culture media, and instruments used are conventional in the art and can be obtained commercially by those skilled in the art unless otherwise specified. The molecular biology experimental procedures such as PCR amplification, enzyme digestion, ligation, and transformation involved in the present invention are conventional in the art or can be performed according to the product instructions of the corresponding reagents unless otherwise specified.

[0032] Streptomyces icebergii ( Streptomyces bingchenggensis ) BC-101-4 is disclosed in the following literature: Wang X, Wang X, Xiang W (2009) Improvement of milbemycin-producing Streptomyces bingchenggensis by rational screening of ultraviolet-and chemically inducedmutants. World J Microbiol Biotechnol 25:1051-1056.

[0033] Streptomyces coelicolor ( Streptomyces coelicolor ) M145 is disclosed in the following literature: Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Foundation, Norwich.

[0034] Streptomyces avermitilis ( Streptomyces avermitilis) S0 is disclosed in the patent with application number CN202310480048.6, and the invention name is a sugar transport protein TP6568 and its application in transforming high-yield Streptomyces.

[0035] Streptomyces crassa Streptomyces rimosus )M4018 is disclosed in the following literature: Yin Shouliang, Lin Zhiwei, Zhang Yuxiu, et al. Engineering of Streptomyces fissiliflorus to improve oxytetracycline production [J]. Chinese Journal of Biotechnology, 2016, 36(7): 72-82.

[0036] Saccharopolyspora spinosa ( Saccharopolyspora spinosa )NRRL 18395 was published in the following literature: Guo Hang, Bai Tingli, Tao Meifeng. Cloning and assembly of rhamnose and forosamine biosynthesis genes from Saccharopolyspora spinosa[J]. Journal of Huazhong Agricultural University, 2012, 31(3): 298-302.

[0037] Streptomyces roseosporus ( Streptomyces roseosporus ) NRRL 11379 is disclosed in the following literature: Yu Jisan, Hong Kui, Lin Haipeng, et al. Optimization of fermentation medium for the production of daptomycin precursor A21978C by Streptomyces roseosporus NRRL11379 [J]. Journal of Anhui Agricultural Sciences, 2008, 36(19): 7974-7976.

[0038] Streptomyces venezuelae ( Streptomyces venezuelae )ISP5230 is disclosed in the following literature: He Jianyong, Yao Xinsheng, LEO.C.VINING. Cloning of chloramphenicol biosynthetic genes in Streptomyces venezuelae [J]. Journal of Shenyang Pharmaceutical University, 2006, 23(11): 731-734.

[0039] Streptomyces albus ( Streptomyces albus )IPPDNR is based on Streptomyces albus Del14 is the starting strain, which heterologously expresses the synthetic gene cluster of daunorubicin. Streptomyces albus Del14 is disclosed in the following literature: MYRONOVSKYI M, ROSENKRäNZER B, NADMID S, et al. Generation of acluster-free Streptomyces albuschassis strains for improved heterologous expression of secondary metabolite clusters [J]. Metabolic Engineering, 2018, 49: 316-24. The synthetic gene cluster of daunorubicin is disclosed in the following literature: Lomovskaya N, Otten SL, Doi-Katayama Y, et al. Doxorubicin overproduction in Streptomyces peucetius :cloning and characterization of the dnU ketoreductase and dnV genes and the doxA cytochrome P-450 hydroxylase gene. J Bacteriol. 1999;181(1):305-318. The construction method of IPPDNR of Streptomyces albus is as follows: an overexpression integration plasmid pSET156-DNR containing the daunorubicin synthesis gene cluster is constructed, the overexpression integration plasmid is transformed into Escherichia coli, and then the overexpression integration plasmid is introduced into the starting strain Del14 through an intergeneric transfer experiment to obtain IPPDNR of Streptomyces albus.

[0040] The above strains were all stored in our laboratory.

[0041] The strain information involved in the following examples is shown in Table 1; the plasmid information involved is shown in Table 2; and the primer information involved is shown in Table 3.

[0042] Table 1 Information on strains involved in the examples

[0043]

[0044] Table 2 Plasmid information involved in the examples

[0045]

[0046] Table 3 Primer information involved in the examples

[0047]

[0048] Example 1: Containing a gene encoding phosphopantetheinyl transferase B3D0 Construction of gene recombinant vector and recombinant bacteria

[0049] In order to improve the production of natural products from actinomycetes, this study mined a PPTase B3D0 (Uniprot protein accession number is A0A2H5B3D0) from the database through bioinformatics analysis and other means. The enzyme is from Kitasatospora ( Kitasatospora sp.) MMS16-BH015, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the enzyme is shown in SEQ ID NO.2.

[0050] SEQ ID NO.1:

[0051] MVFSLEPAANNPNTPGMFARPIQVRGPDGPWEAVRAAVSRYGSALVHARMDDWLPTDLSDPLIPDQLGPDQVRFRRMRHPTVRARFVASRLMLKHTAGAVLGEPPHALELAYKLGGRPYLRGFEQLDVSLSHTADLLVVGLTRRG WIGVDAELRDRQMVGLGTEAQMCTRHEREELERIAEEQRNTALVRLWTLKEAYSKAIGQGMRFRFTEFGFGPRELQAADVRLPDGSPATGAEWTFRTALLQERYTVSVALYDAGFGEAGDGAHALRVEDGLLAELLGGAADAP*

[0052] SEQ ID NO.2:

[0053] ATGGTCTTCTCGCTGGAACCCGCGGCCAACCCGAACACGCCGGGGATGTTCGCCCGCCCGATCCAGGTGCGCGGACCGGACGGGCCGTGGGAGGCCGTGCGCGCGGCGGTGTCCCGGTACGGCAGCGCGCTGGTCCACGCCCGGATGGACGACTGGCTGCCGACGGACCTGTCGGACCCGCTGATCCCGGATCAGCTCGGGCCCGACCAGGTGCGGTTCCGCCGGATGCGCCACCCGACCGTCCGGGCGCGCTTCGTGGCCTCCCGGCTGATGCTCAAGCACACCGCCGGCGCGGTCCTCGGCGAGCCGCCGCACGCCCTGGAACTGGCCTACAAGCTGGGCGGCCGCCCGTACCTGCGCGGTTTCGAACAGCTGGACGTCAGCCTCAGCCACACCGCCGACCTGCTGGTGGTCGGGCTGACCAGGCGCGGCTGGATCGGCGTGGACGCCGAACTCCGTGACCGGCAGATGGTGGGCCTGGGCACCGAGGCCCAGATGTGCACCCGGCACGAGCGGGAGGAGCTGGAGCGGATCGCCGAGGAGCAGCGCAACACCGCGCTGGTGCGCCTGTGGACACTCAAGGAGGCGTACAGCAAGGCCATCGGCCAGGGCATGCGGTTCCGCTTCACCGAGTTCGGCTTCGGGCCGCGGGAGCTCCAGGCGGCGGACGTCCGACTGCCCGACGGCTCCCCCGCGACCGGCGCGGAGTGGACCTTCCGCACCGCCCTGCTGCAGGAGCGCTACACGGTGAGCGTCGCCCTGTACGACGCGGGCTTCGGGGAGGCGGGTGACGGGGCGCACGCGCTGCGGGTGGAGGACGGCCTGCTGGCCGAGCTGCTCGGTGGGGCCGCCGACGCCCCTTGA

[0054] (1) Construction of recombinant vector containing PPTase B3D0 gene

[0055] The gene encoding PPTase B3D0 provided by the present invention was synthesized by Qingke Biotechnology Co., Ltd., and the His-tagged PPTase B3D0 protein expression vector pSET28a-B3D0 was constructed using the integration vector pSET152. B3D0 The gene was overexpressed and the constitutive promoter PermE* was selected as the promoter. The plasmid pSET152-PermE* was double-digested with restriction enzymes XbaI and EcoRI to obtain the linear vector backbone LpSET152-1. PPTase was amplified using primers B3D0-F / R using pSET28a-B3D0 as a template. B3D0 Gene fragments with homology arms were assembled using the Gibson method. B3D0 The gene fragment was assembled with the linear vector backbone LpSET152-1 to obtain a complete plasmid, which was verified by electrophoresis and sequencing to obtain the plasmid pSET152-PermE*-B3D0 (see Figure 1 ).

[0056] (2) Contains PPTase B3D0 Construction of gene-recombinant bacteria

[0057] B3D0 Construction of gene overexpression recombinant bacteria:

[0058] The recombinant plasmid pSET152-PermE*-B3D0 was transformed into Escherichia coli ET12567 (pUZ8002), and then the plasmid was introduced into Streptomyces coelicolor M145, Streptomyces icebergii BC-101-4, Streptomyces avermitilis S0, Saccharopolyspora spinosa NRRL 18395, Streptomyces crassa M4018, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ISP5230 and Streptomyces albus IPPDNR through inter-genus conjugative transfer experiments. The corresponding PPTase was obtained after resistance verification and PCR verification. B3D0 Gene overexpression strains (M145-B3D0, BC-101-4-B3D0, S0-B3D0, NRRL 18395-B3D0, M4018-B3D0, NRRL11379-B3D0, ISP5230-B3D0, and IPPDNR-B3D0).

[0059] Construction of control recombinant bacteria containing empty vector:

[0060] The empty vector pSET152 was introduced into Escherichia coli ET12567 / pUZ8002 by transformation. Then, using the same inter-genus conjugative transfer method, pSET152 was introduced into Streptomyces coelicolor M145, Streptomyces glacialis BC-101-4, Streptomyces avermitilis S0, Saccharopolyspora spinosa NRRL 18395, Streptomyces truncatula M4018, Streptomyces roseospora NRRL 11379, Streptomyces venezuelae ISP5230, and Streptomyces albus IPPDNR, respectively, to obtain the control strains Streptomyces coelicolor M145-C, Streptomyces glacialis BC-101-4-C, Streptomyces avermitilis S0-C, Saccharopolyspora spinosa NRRL 18395-C, Streptomyces truncatula M4018-C, Streptomyces roseospora NRRL 11379-C, Streptomyces venezuelae ISP5230-C, and Streptomyces albus IPPDNR-C.

[0061] Example 2: Verification of the in vitro catalytic effect of PPTase B3D0

[0062] (1) Protein expression and purification

[0063] The plasmid pSET28a-B3D0 in Example 1 was transformed into BL21, induced for expression and then purified to obtain the purified protein B3D0; the first ACP in the synthesis of milbemycin was selected as the substrate protein for catalysis, and the amino acid sequence of ACP is shown in SEQ ID NO.5.

[0064] SEQ ID NO.5:

[0065] GVEAAKVLEDVAGADAPGHGIAEQEHFVTSGFDSAAAVALRNRLNDATGLLLPFTLAFDHPTPAAVADHLHSRL

[0066] The corresponding ACP gene was amplified from the gene cluster of Streptomyces bingchengensis BC-101-4 using primers ACP-F / R. The protein expression plasmid was constructed and then transferred into BL21. After induction of expression, the purified protein ACP was obtained by purification.

[0067] The specific steps for protein induction expression and purification are as follows:

[0068] ① Transform the constructed recombinant protein expression plasmid into Escherichia coli BL21 and culture overnight at 37°C and 250 rpm;

[0069] ②Pick a single clone and place it in 4 mL of LB liquid medium, add the corresponding antibiotics, and culture at 37°C and 250 rpm;

[0070] ③ Transfer the overnight bacterial solution to 250 mL of LB liquid medium at a 1% inoculum volume and culture at 37°C and 250 rpm until the bacterial solution OD reaches 600nm The value is in the range of 0.4-0.6, which is the logarithmic growth period;

[0071] ④Add appropriate amount of IPTG for induction and culture overnight at 16°C and 200 rpm;

[0072] ⑤ Centrifuge at 4000 rpm for 10 min to collect the bacteria;

[0073] ⑥ Resuspend the cells with an appropriate amount of Binding Buffer and disrupt them with ultrasound;

[0074] ⑦ Centrifuge at 6000 rpm at 4°C for 30 min to obtain the supernatant and precipitate. Take a small amount of each, add 5× Loading Buffer to resuspend, boil the sample in boiling water for 10 min, and run SDS-PAGE to detect protein expression.

[0075] ⑧Resuspend the Ni column with Binding Buffer, pass the supernatant through the column, and drain. Repeat twice. Use different concentrations of Elution Buffer (30 mM, 60 mM, 100 mM, 150 mM, 250 mM, and 500 mM) to pass the column, elute the target protein, and collect the flow-through. Take a small amount of the eluate and resuspend it in 5× Loading Buffer. Boil the sample in boiling water for 10 min and run it on SDS-PAGE to detect protein expression.

[0076] The results of SDS-PAGE gel analysis of the purified protein ACP are as follows: Figure 2 As shown in a, the SDS-PAGE gel results of the purified protein B3D0 are as follows Figure 2 As shown in b, it can be seen from these two figures that the target proteins ACP and B3D0 were successfully obtained.

[0077] (2) In vitro reaction and HPLC detection

[0078] The purified and concentrated target protein was dialyzed in dialysate overnight, and the dialyzed ACP and PPTase B3D0 were added to the prepared dialysis buffer containing CoA, Mg 2+ In the in vitro reaction solution, the final concentrations of ACP and PPTase B3D0 were 200 μM and 20 μM, respectively. The reaction was incubated at 25°C for 30 min and quenched on dry ice. Unreacted ACP was used as a control, and the molecular weight of ACP was measured by HPLC and SDS-PAGE to confirm the progress of the reaction.

[0079] HPLC analysis was performed on 50 μL of the purified ACP and the in vitro reaction solution, respectively. Protein detection was performed using a C18 reverse-phase column with a two-phase mobile phase: phase A was water containing 0.1% trifluoroacetic acid, and phase B was acetonitrile. The detection wavelength was 220 nm. The detection time was 20 minutes, with the phase B concentration ranging from 0% to 75% in the first 15 minutes and from 75% to 100% between 15 and 20 minutes.

[0080] The results of HPLC detection of the prosthetic group status of ACP are as follows Figure 2 As shown in c in the figure, it can be seen that B3D0 can achieve partial prosthetic group formation of ACP.

[0081] Example 3: Application of PPTase B3D0 in increasing the yield of type I polyketide milbemycins in Streptomyces bingchengensis

[0082] The recombinant strains BC-101-4-B3D0 and BC-101-4-C of Streptomyces bingchengensis obtained in Example 1, as well as the starting strain BC-101-4, were inoculated onto SKYM solid culture medium and cultured at 28°C for 9 days. Approximately 1 square centimeter of spores were scraped and inoculated into Streptomyces bingchengensis seed culture medium and cultured at 28°C and 250 rpm for 46 hours. Subsequently, a 6% inoculum was inoculated into Streptomyces bingchengensis fermentation medium and cultured at 28°C and 250 rpm for 9 days.

[0083] The spore production medium was SKYM medium, which was composed of 4 g / L sucrose, 1 g / L skim milk powder, 2 g / L yeast extract powder, 5 g / L malt extract powder, 20 g / L agar powder, and the balance was water.

[0084] The composition of the seed culture medium is as follows: 5 g / L yeast extract powder, 10 g / L sucrose, 1 g / L skim milk powder, 3.5 g / L bacterial peptone, 0.5 g / L potassium hydrogen phosphate, and the balance is water.

[0085] The composition of the fermentation medium was as follows: 80 g / L sucrose, 20 g / L soybean cake powder, 1 g / L skimmed milk powder, 0.1 g / L ferrous sulfate, 1 g / L potassium dihydrogen phosphate, 3 g / L calcium carbonate, and the balance was water.

[0086] Milbemycin detection method: 0.5 mL of the fermentation broth of Streptomyces bingchengensis was collected and thoroughly mixed with 1.5 mL of ethanol. The treated sample was shaken and turned for 30 minutes to extract the milbemycin in the fermentation broth. The preliminarily treated sample was then centrifuged at 12,000 rpm for 15 minutes. The upper milbemycin extract was aspirated and passed through a 0.22 μm organic filter membrane for liquid chromatography detection. The detection instrument was an Agilent high-performance liquid chromatography (HPLC), the chromatographic column was a C18 column (Zorbax, 4.6 mm × 250 mm, 5 μm), the detection wavelength was 242 nm, the injection volume was 20 μL, and the mobile phase flow rate was 1.0 mL / min. The specific chromatographic conditions were as follows: mobile phase A (methanol) was eluted from 0% to 100% within 0-15 minutes, and mobile phase B (volume ratio of acetonitrile: methanol: water = 7:2:1) was gradient eluted from 100% to 0%; 15-17 minutes, mobile phase A was maintained at 100%; 17-25 minutes min, mobile phase A (methanol) was eluted from 100% to 0%, and mobile phase B was gradient eluted from 0% to 100%; 25-27 min, mobile phase B was maintained at 100%, and the column temperature was 28°C.

[0087] The results of milbemycin production test are as follows Figure 3 As shown, compared with the starting strain BC-101-4, the overexpression B3D0 The recombinant strain BC-101-4-B3D0 increased milbemycin production by 43.41% to 1398.63 mg / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type I polyketide production, which is of great significance for constructing high-yielding actinomycete strains and promoting the industrialization of natural products.

[0088] Example 4: Application of PPTase B3D0 in increasing the yield of polyketide avermectins of Streptomyces avermitilis type I

[0089] The recombinant Streptomyces avermitilis strains S0-B3D0 and S0-C obtained in Example 1 and the starting strain S0 were inoculated onto YMS solid medium and cultured at 28°C for 6 days. An approximately 1 cm² bacterial disc was taken using an inoculation shovel and inoculated into a Streptomyces avermitilis seed culture medium and cultured at 28°C, 250 rpm, for 40 h. Subsequently, a 6% inoculum was inoculated into a Streptomyces avermitilis fermentation medium and cultured at 28°C, 250 rpm, for 10 days.

[0090] The conidial medium was YMS medium, which had the following composition: 4 g / L soluble starch, 4 g / L yeast extract powder, 10 g / L malt extract powder, 20 g / L agar powder, and the balance was water.

[0091] The composition of the seed culture medium is as follows: 25 g / L corn starch, 2 g / L soybean cake powder, 15 g / L peanut cake powder, 4 g / L yeast extract powder, 0.026 g / L cobalt chloride hexahydrate, 0.03 g / L α-amylase, and the balance is water.

[0092] The composition of the fermentation medium was as follows: 120 g / L corn starch, 38 g / L soybean cake powder, 0.8 g / L calcium carbonate, 8 g / L yeast extract, 0.02 g / L cobalt chloride hexahydrate, 0.1 g / L α-amylase, 0.3 g / L ammonium sulfate, 0.022 g / L sodium molybdate monohydrate, 0.0023 g / L manganese sulfate monohydrate, and the balance was water.

[0093] Abamectin detection method: 0.25 mL of the fermentation broth or fermentation supernatant of Streptomyces avermitilis was thoroughly mixed with 1.20 mL of methanol. The sample was ultrasonically treated for 40 minutes to extract total and extracellular avermectins from the fermentation broth. The pre-treated sample was then centrifuged at 12,000 rpm for 10 minutes. The supernatant avermectin extract was aspirated and filtered through a 0.22 μm organic filter membrane before liquid chromatography (HPLC) analysis. The detection instrument was a Shimadzu LC HPLC system using a C18 column (Zorbax, 4.6 mm × 250 mm, 5 μm). The detection wavelength was 246 nm, the injection volume was 20 μL, the mobile phase was 90% methanol, the flow rate was 1.0 mL / min, and the column temperature was 35°C.

[0094] The test results of avermectin production are as follows Figure 4 As shown, compared with the starting strain S0, the overexpression B3D0 The recombinant strain S0-B3D0 increased avermectin production by 28.17% to 2.73 g / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type I polyketide production, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0095] Example 5: Application of PPTase B3D0 in increasing the yield of polyketide spinosad from Saccharopolyspora spinosa type I

[0096] The recombinant Saccharopolyspora spinosa strains NRRL 18395-B3D0, NRRL 18395-C, and the starting strain NRRL 18395 obtained in Example 1 were inoculated onto YD solid medium and cultured at 28°C for 6 days. Spores were collected with water to prepare a spore suspension, and a corresponding volume of the spore suspension was inoculated into 50 mL of seed medium at a 1% inoculum size and cultured at 28°C, 250 rpm, for 3 days. The seed culture solution in logarithmic phase growth was transferred to fermentation medium at a 2% inoculum size and cultured at 28°C, 250 rpm, for 10 days.

[0097] The sporulation medium was YD medium, which had the following composition: 4 g / L glucose, 4 g / L yeast extract, 10 g / L maltose, 20 g / L agar powder, 1.5 g / L calcium chloride, 2 g / L magnesium chloride, and the balance was water.

[0098] The composition of the seed culture medium was as follows: 30 g / L tryptic soy broth, 3 g / L yeast extract, 10 g / L glucose, 2 g / L magnesium sulfate, and the balance was water.

[0099] The composition of the fermentation medium was as follows: 50 g / L glucose, 20 g / L dextrin, 20 g / L cottonseed peptone, 20 g / L sodium chloride, 1 g / L potassium hydrogen phosphate trihydrate, 0.05 g / L ferrous sulfate heptahydrate, 1 g / L calcium carbonate, and the balance was water.

[0100] Spinosad detection method: 5 mL of fermentation broth was thoroughly mixed with an equal volume of methanol and sonicated for 30 minutes to extract total and extracellular spinosyns. The mixture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm microporous filter and then analyzed by liquid chromatography. The detection instrument was a Shimadzu HPLC (HPLC-LC) with a reversed-phase C18 column. The mobile phase was a 40:50:10 volume ratio of methanol:acetonitrile:water at a flow rate of 0.5 mL / min, the column temperature was 25°C, the injection volume was 10 μL, and the detection wavelength was 246 nm.

[0101] The results of the spinosad production test are as follows Figure 5 As shown, compared with the starting strain NRRL 18395, the overexpression B3D0 The recombinant strain NRRL 18395-B3D0 increased spinosad production by 30.21% to 35.42 mg / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type I polyketide production, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0102] Example 6: Application of PPTase B3D0 in increasing the yield of polyketide actinomycetamine from Streptomyces coelicolor type II

[0103] The recombinant strains M145-B3D0 and M145-C of Streptomyces coelicolor obtained in Example 1 and the original strain M145 were inoculated on MS solid medium and cultured at 28°C for 5 days. Spores were collected and the spore concentration was 4×10 6 The inoculum of 1000 μg / mL was transferred to the fermentation medium of Streptomyces coelicolor, and 0.5 mL of the fermentation liquid was taken after culturing at 28°C and 250 rpm for 5 days.

[0104] The spore production medium was MS medium, which had the following composition: 20 g / L soybean cake powder, 20 g / L mannitol, 20 g / L agar powder, and the balance was water.

[0105] The composition of the fermentation medium: 50 g / L PEG6000, 1.23 g / L magnesium sulfate heptahydrate, 10 g / L glucose, 2 g / L acid-hydrolyzed casein, 1 mL / L potassium hydrogen phosphate trihydrate, 25 mM 5×TES buffer, 1 mM sodium dihydrogen phosphate, 1 mM potassium hydrogen phosphate, trace elements: ZnSO4·7H2O, NaCl, FeSO4·7H2O, MnCl2·4H2O, CaCl2·6H2O 0.1 g / L each, and the balance is water.

[0106] Actinomycin detection method: Treat with 0.5 mL of 1 M NaOH, centrifuge, and measure the OD of the supernatant 608nm , detect the production of actinomycetin (Act).

[0107] The results of the actinomycin production test are as follows Figure 6 As shown, compared with the starting strain M145, the overexpression B3D0 The recombinant strain M145-B3D0 increased actinomycetin production by 76.47%, reaching 218.42 mg / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type II polyketide production, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0108] Example 7: Application of PPTase B3D0 in increasing the yield of polyketide oxytetracycline type II in Streptomyces pyrifera

[0109] The recombinant strains M4018-B3D0 and M4018-C of Streptomyces truncatum obtained in Example 1 and the original strain M4018 were inoculated on MS solid medium, cultured at 30°C for 5 days, and then spores were scraped and inoculated into Streptomyces truncatum seed medium. The culture was placed at 30°C and 250 rpm for 24 h, and then transferred to Streptomyces truncatum fermentation medium with an inoculum size of 5% and cultured at 28°C and 250 rpm for 8 days.

[0110] The spore production medium was MS medium, which had the following composition: 20 g / L soybean cake powder, 20 g / L mannitol, 20 g / L agar powder, and the balance was water.

[0111] The composition of the seed culture medium is as follows: 30 g / L starch, 3 g / L soybean cake powder, 4 g / L ammonium sulfate, 5 g / L calcium carbonate, 4 g / L corn steep liquor, 5 g / L sodium chloride, 0.15 g / L potassium dihydrogen phosphate, and the balance is water.

[0112] The composition of the fermentation medium is as follows: 150 g / L starch, 20 g / L soybean cake powder, 14 g / L ammonium sulfate, 14 g / L calcium carbonate, 4 g / L corn steep liquor, 4 g / L sodium chloride, 0.1 g / L potassium dihydrogen phosphate, 10 μg / L cobalt chloride, 0.1%-0.2% amylase, and the balance is water.

[0113] Detection method for oxytetracycline: Take 1.5 mL of fermentation broth, acidify it with 9 mol / L hydrochloric acid to a pH of 1.5 to 1.7, centrifuge, take the supernatant and pass it through a membrane for HPLC detection. Detection method: The detection instrument is Agilent high performance liquid chromatography (HPLC), using an SB-C18 (250 mm×4.6 mm, Agilent, America) column, the volume ratio of the mobile phase is water: methanol: acetonitrile: 2 mM phosphoric acid solution = 60:10:20:10, the flow rate is 1.0 mL / min, the detection wavelength is 350 nm, and the injection volume is 10 μL.

[0114] The test results of oxytetracycline production are as follows Figure 7 As shown, compared with the starting strain M4018, the overexpression B3D0 The recombinant strain M4018-B3D0 increased oxytetracycline production by 69.90% to 2.07 g / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type II polyketide production, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0115] Example 8: Application of PPTase B3D0 in increasing the yield of polyketide daptomycin of Streptomyces venezuelae type II

[0116] The recombinant strains ISP5230-B3D0 and ISP5230-C of Streptomyces venezuelae obtained in Example 1 and the original strain ISP5230 were inoculated on MYM solid medium, cultured at 30°C for 5 days, and then spores were scraped and inoculated into seed medium. After shaking culture at 28°C for 20 hours, the spores were transferred to fermentation medium and continued to be shaken at 28°C for 48 hours.

[0117] The spore production medium was MYM medium, which had the following composition: 10 g / L malt extract powder, 4 g / L yeast extract powder, 4 g / L maltose, 20 g / L agar powder, and the balance was water.

[0118] The seed culture medium was liquid MYM medium.

[0119] The fermentation medium consisted of MSM liquid medium, glucose, and phosphate buffer stock solutions, with a final glucose concentration of 33 mM and a final phosphate buffer concentration of 50 μM. Each liter of MSM liquid medium (pH 7.5) contained 0.4 g of MgSO4, 1.9 g of MOPS, 9 mL of saline solution (an aqueous solution containing 1 g / 100 mL NaCl and 1 g / 100 mL CaCl2), 4.5 mL of a 0.2 g / 100 mL FeSO4·7H2O aqueous solution, 4.5 mL of a trace element solution, and 7.8 g of L-isoleucine. Each liter of the trace element solution contained 880 mg of ZnSO4·7H2O, 39 mg of CuSO4·5H2O, 6.1 mg of MnSO4·4H2O, 5.7 mg of H3BO3, and 3.7 mg of (NH4)6Mo7O. 24 4H2O. Each liter of phosphate buffer stock solution (9 mM) contains 10.5 g K2HPO4 and 4.5 g KH2PO4.

[0120] Jedomycin detection method: Take 1 mL of fermentation broth, centrifuge and filter to remove the bacteria, add an equal volume of ethyl acetate for extraction, and take the upper organic phase; evaporate the extract to dryness in a fume hood at room temperature, dissolve it in a certain volume of methanol or DMSO, and perform HPLC detection. Detection method: The detection instrument is Agilent high-performance liquid chromatography (HPLC), using an SB-C18 (250 mm×4.6 mm, Agilent, America) column, the mobile phase is A: water, B: acetonitrile + 0.1% trifluoroacetic acid, gradient elution, flow rate, 1.0 mL / min, detection wavelength 316 nm, and injection volume 20 μL.

[0121] The results of the jedomycin production test are as follows Figure 8 As shown, compared with the starting strain ISP5230, the overexpression B3D0The recombinant strain ISP5230-B3D0 increased jedomycin production by 39.04% to 334.99 μg / mL. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type II polyketide production, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0122] Example 9: Application of PPTase B3D0 in increasing the yield of polyketide daunorubicin type II of Streptomyces albus

[0123] The recombinant strains IPPDNR-B3D0 and IPPDNR-C of Streptomyces albus obtained in Example 1 and the original strain IPPDNR were inoculated on MS solid medium, cultured at 30°C for 3 days, and 1 cm × 2 cm spores were scraped and inoculated into a primary seed medium (Tryptone Soy Broth TSB). The culture was carried out at 30°C and 220 rpm for 30 hours, and then transferred to a secondary seed medium at a 5% inoculum size and cultured at 30°C and 220 rpm for 24 hours. The culture was then transferred to a fermentation medium at a 10% inoculum size and cultured at 30°C and 250 rpm for 10 days.

[0124] The spore production medium was MS medium, which had the following composition: 20 g / L soybean cake powder, 20 g / L mannitol, 20 g / L agar powder, and the balance was water.

[0125] The composition of the secondary seed culture medium is as follows: 1 g / L yeast extract, 4 g / L glucose, 3 g / L soybean powder, 0.2 g / L calcium carbonate, and the balance is water.

[0126] The composition of the fermentation medium was as follows: 4 g / L glucose, 0.4 g / L yeast extract, 1 g / L malt extract, 5 g / L dextrin, 5 mL / L soybean oil, and the balance was water.

[0127] Daunorubicin detection method: Take 1 mL of fermentation broth, add 9 mL of methanol, let it stand for 12 hours, then centrifuge, take the supernatant and pass it through a membrane for HPLC detection. Detection method: The detection instrument is Agilent high performance liquid chromatography (HPLC), using an SB-C18 (250 mm×4.6 mm, Agilent, America) column, mobile phase A: 0.01 M ammonium acetate aqueous solution (pH=4.2), mobile phase B: acetonitrile, flow rate 1.0 mL / min, detection wavelength is 254 nm, column temperature 37°C, gradient elution, injection volume 20 μL.

[0128] The results are as follows Figure 9 As shown, compared with the starting strain IPPDNR, overexpression B3D0The recombinant strain IPPDNR-B3D0 increased daunorubicin production by 27.27%, reaching 33.73 μg / mL. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing type II polyketide production, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0129] Example 10: Application of PPTase B3D0 in increasing the yield of the polyether natural product Nanchangmycin from Streptomyces bingchengensis

[0130] The recombinant strains BC-101-4-B3D0 and BC-101-4-C of Streptomyces bingchengensis obtained in Example 1, as well as the starting strain BC-101-4, were inoculated onto SKYM solid medium and cultured at 28°C for 9 days. Approximately 1 square centimeter of spores were scraped and inoculated into Streptomyces bingchengensis seed culture medium and cultured at 28°C, 250 rpm, for 46 hours. Subsequently, a 6% inoculum was inoculated into Streptomyces bingchengensis fermentation medium and cultured at 28°C, 250 rpm, for 9 days.

[0131] The spore production medium was SKYM medium, which was composed of 4 g / L sucrose, 1 g / L skim milk powder, 2 g / L yeast extract powder, 5 g / L malt extract powder, 20 g / L agar powder, and the balance was water.

[0132] The composition of the seed culture medium is as follows: 5 g / L yeast extract powder, 10 g / L sucrose, 1 g / L skim milk powder, 3.5 g / L bacterial peptone, 0.5 g / L potassium hydrogen phosphate, and the balance is water.

[0133] The composition of the fermentation medium was as follows: 80 g / L sucrose, 20 g / L soybean cake powder, 1 g / L skimmed milk powder, 0.1 g / L ferrous sulfate, 1 g / L potassium dihydrogen phosphate, 3 g / L calcium carbonate, and the balance was water.

[0134] Nanchangmycin detection method: 0.5 mL of the fermentation broth of Streptomyces bingchengensis was collected and thoroughly mixed with 1.5 mL of ethanol. The treated sample was flipped and shaken for 30 minutes to extract nanchangmycin in the fermentation broth. The preliminarily treated sample was then centrifuged at 12,000 rpm for 15 minutes. The upper layer of nanchangmycin extract was aspirated and passed through a 0.22 μm organic filter membrane for liquid phase detection. The detection instrument was an Agilent high performance liquid chromatography (HPLC), the chromatographic column was a C18 column (Zorbax, 4.6 mm × 250 mm, 5 μm), the detection wavelength was 242 nm, the injection volume was 20 μL, the mobile phase flow rate was 1.0 mL / min, and the specific chromatographic conditions were as follows: mobile phase A (methanol) was eluted from 0% to 100% within 0-15 minutes, and the volume ratio of mobile phase B was acetonitrile: methanol: water = 7:2:1, with gradient elution from 100% to 0%; 15-17 minutes, mobile phase A was maintained at 100%; 17-25 min, mobile phase A (methanol) was eluted from 100% to 0%, and mobile phase B was gradient eluted from 0% to 100%; 25-27 min, mobile phase B was maintained at 100%, and the column temperature was 28°C.

[0135] The results of Nanchangmycin production test are as follows Figure 10 As shown, compared with the starting strain BC-101-4, the overexpression B3D0 The recombinant strain BC-101-4-B3D0 increased nanchangmycin production by 45.33%, reaching 1698.29 mg / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing the production of polyether compounds, which is of great significance for constructing high-yield actinomycete strains for natural products and promoting the industrialization of natural products.

[0136] Example 11: Application of PPTase B3D0 in increasing the yield of the non-ribosomal peptide natural product daptomycin

[0137] The recombinant strains NRRL 11379-B3D0 and NRRL 11379-C of Streptomyces roseosporus obtained in Example 1 and the original strain NRRL 11379 were inoculated on DAI solid medium, cultured at 28°C for 7 days, and then the spores were scraped and inoculated into a primary seed medium of Streptomyces roseosporus. The culture was placed at 28°C and 250 rpm for 60 h, and then transferred to a secondary seed medium with an inoculum size of 5%, cultured at 28°C and 250 rpm for 36 h, and then transferred to a fermentation medium of Streptomyces roseosporus with an inoculum size of 5%, and cultured at 28°C and 250 rpm for 10 days. Starting at 48 h, 0.5 mL of 2% (W / V) sterile sodium decanoate solution was added every 12 h.

[0138] The spore production medium was DA1 medium, which had the following composition: 4 g / L glucose, 4 g / L yeast extract powder, 10 g / L malt extract powder, 2 g / L calcium carbonate, 20 g / L agar powder, and the balance was water.

[0139] The composition of the seed culture medium is as follows: 5 g / L glucose, 15 g / L dextrin, 5 g / L bacterial peptone, 5 g / L yeast extract powder, 5 g / L peanut powder, 0.5 g / L potassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium carbonate, and the balance is water.

[0140] The composition of the fermentation medium was as follows: 50 g / L soluble starch, 10 g / L glucose, 10 g / L dextrin, 10 g / L tryptone, 0.3 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium carbonate, and the balance was water.

[0141] Daptomycin detection method: Take 1 mL of fermentation broth, centrifuge twice at 13,000 rpm and 4°C for 15 minutes, take the supernatant and pass it through a membrane for HPLC detection. Detection method: The detection instrument is Agilent high-performance liquid chromatography (HPLC), using an SB-C18 (250 mm×4.6 mm, Agilent, America) column, the volume ratio of the mobile phase is water:acetonitrile = 56:44, the flow rate is 1.0 mL / min, the detection wavelength is 218 nm, and the injection volume is 20 μL.

[0142] The results of daptomycin production test are as follows Figure 11 As shown, compared with the starting strain NRRL 113792, the overexpression B3D0 The recombinant strain NRRL 11379-B3D0 increased daptomycin production by 71.98% to 389.43 mg / L. This suggests that the discovered PPTase element B3D0 is an effective biosynthetic element capable of increasing the production of non-ribosomal peptide compounds, which is of great significance for constructing high-yielding strains of actinomycetes and promoting the industrialization of natural products.

[0143] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of a gene encoding phosphopantetheinyltransferase B3D0 in promoting the synthesis of natural products, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO.2, and the natural product is a polyketide compound, a polyether compound or a non-ribosomal peptide compound; the polyketide compound is milbemycin, avermectin, spinosad, actinomycin, oxytetracycline or daptomycin, the polyether compound is nanchangmycin, and the non-ribosomal peptide compound is daptomycin; the application is in Streptomyces bingchengensis ( Streptomyces bingchenggensis ) BC-101-4 to overexpress the encoding gene to promote the synthesis of milbemycin or nanchangmycin, or in Streptomyces avermitilis ( Streptomyces avermitilis ) S0 to overexpress the coding gene to promote the synthesis of avermectin, or in Saccharopolyspora spinosa ( Saccharopolyspora spinosa ) NRRL 18395 to promote the synthesis of spinosad, or in Streptomyces coelicolor ( Streptomyces coelicolor ) M145 to promote the synthesis of actinomycetine, or in Streptomyces pyrrogenes ( Streptomyces rimosus ) M4018 to promote the synthesis of oxytetracycline, or in Streptomyces venezuelae ( Streptomyces venezuelae ) ISP5230 to promote the synthesis of degamycin, or in Streptomyces roseosporus ( Streptomyces roseosporus ) NRRL 11379 overexpressed the encoding gene to promote the synthesis of daptomycin.

2. Use of a recombinant vector containing the coding gene according to claim 1 in promoting the synthesis of natural products, characterized in that: The natural product is a polyketide compound, a polyether compound or a non-ribosomal peptide compound; the polyketide compound is milbemycin, avermectin, spinosad, actinomycin, oxytetracycline or daptomycin, the polyether compound is nanchangmycin, and the non-ribosomal peptide compound is daptomycin; the application is to introduce the recombinant vector into Streptomyces bingchengensis BC-101-4 to promote the synthesis of milbemycin or nanchangmycin, or to introduce the recombinant vector into Streptomyces avermectin S0 to promote the synthesis of avermectin, or to introduce the recombinant vector into Saccharopolyspora spinosa NRRL 18395 to promote the synthesis of spinosad, or to introduce the recombinant vector into Streptomyces coelicolor M145 to promote the synthesis of actinomycin, or to introduce the recombinant vector into Streptomyces crassa M4018 to promote the synthesis of oxytetracycline, or to introduce the recombinant vector into Streptomyces venezuelae ISP5230 to promote the synthesis of daptomycin, or to introduce the recombinant vector into Streptomyces roseosporus NRRL 11379 to promote the synthesis of daptomycin.

3. The use according to claim 2, characterized in that The method for constructing the recombinant vector comprises the following steps: constructing a recombinant expression vector pSET28a-B3D0 containing a gene encoding phosphopantetheinyl transferase B3D0, performing double enzyme digestion on a plasmid pSET152-PermE* carrying a constitutive promoter PermE* using two restriction endonucleases, XbaI and EcoRI, to obtain a linear vector skeleton LpSET152-1; amplifying the pSET28a-B3D0 gene using a template. B3D0 Gene fragments with homology arms were assembled using the Gibson method. B3D0 The gene fragment was assembled with the linear vector backbone LpSET152-1 to obtain the recombinant vector pSET152-PermE*-B3D0.

4. The use according to claim 3, characterized in that The amplification B3D0 The primers used for the gene fragment are the upstream primer B3D0-F having a nucleotide sequence as shown in SEQ ID NO.4 and the downstream primer B3D0-R having a nucleotide sequence as shown in SEQ ID NO.

3.

5. Use of a recombinant bacterium containing the coding gene of claim 1 or the recombinant vector of claim 2 in promoting the synthesis of natural products, characterized in that: The natural product is a polyketide compound, a polyether compound or a non-ribosomal peptide compound; the polyketide compound is milbemycin, avermectin, spinosad, actinomycin, oxytetracycline or daptomycin, the polyether compound is nanchangmycin, and the non-ribosomal peptide compound is daptomycin; in the application, when the starting strain of the recombinant bacteria is Streptomyces bingchengensis BC-101-4, the recombinant bacteria is used to promote the synthesis of milbemycin or nanchangmycin; when the starting strain of the recombinant bacteria is Streptomyces avermitilis S0, the recombinant bacteria is used to promote the synthesis of avermectin; when the starting strain of the recombinant bacteria is Saccharopolyspora spinosa NRRL 18395, the recombinant bacteria is used to promote the synthesis of spinosad; when the starting strain of the recombinant bacteria is Streptomyces coelicolor M145, the recombinant bacteria is used to promote the synthesis of actinomycin; when the starting strain of the recombinant bacteria is Streptomyces crassa M4018, the recombinant bacteria is used to promote the synthesis of oxytetracycline; when the starting strain of the recombinant bacteria is Streptomyces venezuelae ISP5230, the recombinant bacteria is used to promote the synthesis of daptomycin; when the starting strain of the recombinant bacteria is Streptomyces roseosporus NRRL 11379, the recombinant bacteria is used to promote the synthesis of daptomycin.

6. The use according to claim 5, characterized in that The method for constructing the recombinant bacteria comprises the following steps: constructing an overexpression integration plasmid encoding a phosphopantetheinyltransferase B3D0 gene, transforming the overexpression integration plasmid into Escherichia coli, and then introducing the overexpression integration plasmid into a starting strain through an inter-genus conjugative transfer experiment to obtain the recombinant bacteria.

7. The use according to claim 6, characterized in that The Escherichia coli is Escherichia coli ET12567 carrying plasmid pUZ8002.

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