Phosphoric acid pantetheinyl transferase B3D0, coding gene thereof, recombinant vector containing gene and application of recombinant bacteria

Abstract

The invention discloses phosphopantetheinyl transferase B3D0, a coding gene thereof, a recombinant vector containing the gene and application of recombinant bacteria, and belongs to the technical field of gene engineering. In order to improve the yield of natural products such as polyketones and non-ribosome peptides, the efficient phosphopantetheinyl transferase B3D0 is excavated from a database by means of bioinformatics analysis and the like, the amino acid sequence of the transferase B3D0 is as shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene of the transferase B3D0 is as shown in SEQ ID NO.2. By constructing a recombinant vector and recombinant bacteria containing the encoding gene of the enzyme, the yield of natural products such as polyketone, polyether, non-ribosome peptide and the like can be effectively increased by over-expressing the enzyme.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to applications of phosphopantetheine transferase B3D0, its encoding gene, a recombinant vector containing the gene, and a recombinant bacterium. Background Art

[0002] Polyketide compounds are a large category of natural products. They have 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 production bacteria of polyketide natural product pesticides. However, the metabolic regulatory network of Streptomyces is complex, and there are still several key mechanisms that affect the biosynthesis of natural products such as polyketides that have not been thoroughly analyzed, resulting in slow development of high-yield bacteria. The fermentation industry faces common problems such as low product yield and high production cost, which seriously hinders the development and application of polyketide compounds. It 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 in an inactive apoprotin form ( apo -) into the active full protein form ( Holo -), phosphopantetheinyl transferase (PPTase) is the enzyme that catalyzes this reaction, which transfers the phosphopantetheinyl transfer arm (PPant) of coenzyme A to the serine residue of ACP to complete the activation of ACP. Therefore, PPTase is a key enzyme for PKS to function, and is also a key enzyme for the biosynthesis of natural products such as polyketides and non-ribosomal peptides.

[0004] At present, PPTase is an important catalytic element, and there is little research on efficient PPTase that can be used for strain modification, resulting in a shortage of catalytic elements and a single choice of PPTase elements for efficient synthesis of natural products. Therefore, the discovery of highly efficient PPTase is of great value for increasing the yield of natural products such as polyketides and non-ribosomal peptides. Summary of the invention

[0005] In order to increase the yield of natural products such as polyketides and non-ribosomal peptides, the present invention uses bioinformatics analysis and other means to mine a highly efficient PPTase B3D0 (Uniprot protein accession number is A0A2H5B3D0) from the database, and by constructing a recombinant vector and recombinant bacteria containing the enzyme encoding gene, it is 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: The first object of the present invention is to provide an application of phosphopantetheinyl transferase B3D0 in promoting the synthesis of natural products, wherein the amino acid sequence of the phosphopantetheinyl transferase 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.

[0007] The second object of the present invention is to provide an application 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.

[0008] The third object of the present invention is to provide an application of a recombinant vector containing a phosphopantetheinyl transferase B3D0 encoding gene in promoting the synthesis of natural products, wherein the nucleotide sequence of the phosphopantetheinyl transferase B3D0 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] 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 phosphopantetheine transferase B3D0, performing double restriction 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.

[0010] In one embodiment of the present invention, the amplification B3D0 The primers used for the gene fragment are the upstream primer B3D0-F with a nucleotide sequence as shown in SEQ ID NO.4 and the downstream primer B3D0-R with a nucleotide sequence as shown in SEQ ID NO.3.

[0011] 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.

[0012] In one embodiment of the present invention, the method for constructing the recombinant bacteria comprises the following steps: constructing an overexpression integration plasmid of the phosphopantetheinyltransferase B3D0 encoding gene, transforming the overexpression integration plasmid into Escherichia coli, and then introducing the overexpression integration plasmid into the starting strain through an inter-genus conjugation transfer experiment to obtain the recombinant bacteria.

[0013] In one embodiment of the present invention, the Escherichia coli is ET12567 (pUZ8002).

[0014] 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 fissipes ( 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.

[0015] In one embodiment of the present invention, when the starting strain is Streptomyces 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 fissure 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 roseosporeus 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 jedomycin; when the starting strain is Streptomyces albus IPPDNR, the recombinant bacteria is used to increase the yield of daunorubicin.

[0016] Beneficial effects of the present invention: The present invention utilizes bioinformatics analysis and other means to mine a highly efficient PPTase B3D0 from a database, and obtains a total of 8 high-yield recombinant strains carrying the highly efficient PPTase element B3D0 by constructing a recombinant vector and recombinant bacteria containing the enzyme encoding gene. The actinomycin 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 production of nanchangmycin 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 spinosad 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 excavated 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

[0017] Figure 1 For gene B3D0 Schematic diagram of the construction of the overexpression integration plasmid pSET152-PermE*-B3D0; Figure 2 The result of the in vitro catalytic reaction of PPTase B3D0 is shown in FIG. Figure 2a in the figure is the SDS-PAGE gel run result of the purified substrate ACP, M is the 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 the 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; Figure 3 It is a graph showing the detection 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; Figure 4 It is a graph showing the test results of the avermectin production of the recombinant strains S0-B3D0, S0-C and the starting strain S0 of Streptomyces avermitilis; Figure 5 The graph is a test result of the spinosad production of the recombinant Saccharopolyspora strains NRRL 18395-B3D0, NRRL 18395-C and the starting strain NRRL18395; Figure 6 The figure is the test result of the actinomycetoma rhodamine production of the recombinant strains M145-B3D0, M145-C and the starting strain M145 of Streptomyces coelicolor; Figure 7 The graph is a test result of oxytetracycline production of the recombinant strains M4018-B3D0, M4018-C and the starting strain M4018 of Streptomyces fissiliflorus; Figure 8 It is the test result of the yield of jedomycin of the recombinant strains ISP5230-B3D0, ISP5230-C and the starting strain ISP5230 of Streptomyces venezuelae; Fig. 9 It is a graph showing the detection results of daunorubicin production of the recombinant strains IPPDNR-B3D0, IPPDNR-C of Streptomyces albus and the starting strain IPPDNR; Fig.10 The figure is the test result of the yield of Nanchangmycin of the recombinant strains BC-101-4-B3D0, BC-101-4-C and the original strain BC-101-4 of Streptomyces bingchengensis; Fig.11 This is a graph showing the test results of daptomycin production by the recombinant strains of Streptomyces roseosporus NRRL 11379-B3D0, NRRL 11379-C and the original strain NRRL11379. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. 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 part of the embodiments of the present invention rather than all the embodiments. In this field, if other technicians do not make creative work, the embodiments they obtain are protected by the present invention.

[0019] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels. The molecular biology experimental operations such as PCR amplification, enzyme ligation, transformation, etc. involved in the present invention are conventional experimental operations in the art or can be performed according to the product instructions of the corresponding reagents unless otherwise specified.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Streptomyces fissilifolius 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.

[0024] Saccharopolyspora spinosa Saccharopolyspora spinosa ) NRRL 18395 was published in the following document: Guo Hang, Bai Tingli, Tao Meifeng. Cloning and assembly of rhamnose and forosamine biosynthesis genes in Saccharopolyspora spinosa[J]. Journal of Huazhong Agricultural University, 2012, 31(3): 298-302.

[0025] 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 producing daptomycin precursor A21978C by Streptomyces roseosporus NRRL11379 [J]. Anhui Agricultural Sciences, 2008, 36(19): 7974-7976.

[0026] Streptomyces venezuelae ( Streptomyces venezuelae )ISP5230 is disclosed in the following document: 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.

[0027] 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 albus chassis 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 oeLhketoreductase 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: construct an overexpression integration plasmid pSET156-DNR containing the daunorubicin synthesis gene cluster, transform the overexpression integration plasmid into Escherichia coli, and then introduce the overexpression integration plasmid into the starting strain Del14 through an inter-genus conjugation transfer experiment to obtain IPPDNR of Streptomyces albus.

[0028] The above strains were stored in our laboratory.

[0029] 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.

[0030] Table 1 Information on strains involved in the examples

[0031] Table 2 Plasmid information involved in the examples

[0032] Table 3 Primer information involved in the examples

[0033] Example 1: Containing a gene encoding phosphopantetheinyl transferase B3D0 Construction of gene recombinant vector and recombinant bacteria 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 Kitasatosporium ( 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.

[0034] SEQ ID NO.1: MVFSLEPAANPNTPGMFARPIQVRGPDGPWEAVRAAVSRYGSALVHARMDDWLPTDLSDPLIPDQLGPDQVRFRRMRHPTVRARFVASRLMLKHTAGAVLGEPPHALELAYKLGGRPYLRGFEQLDVSLSHTADLLVVGLTRRGWIGVDAELRDRQMVGLGTEAQMCTRHEREELERIAEEQRNTALVRLWTLKEAYSKAIGQGMRFRFTEFGFGPRELQAADVRLPDGSPATGAEWTFRTALLQERYTVSVALYDAGFGEAGDGAHALRVEDGLLAELLGGAADAP* SEQ ID NO.2: ATGGTCTTCTCGCTGGAACCCGCGGCCAACCCGAACACGCCGGGGATGTTCGCCCGCCCGATCCAGGTGCGCGGACCGGACGGGCCGTGGGAGGCCGTGCGCGCGGCGGTGTCCCGGTACGGCAGCGCGCTGGTCCACGCCCGGATGGACGACTGGCTGCCGACGGACCTGTCGGACCCGCTGATCCCGGATCAGCTCGGGCCCGACCAGGTGCGGTTCCGCCGGATGCGCCACCCGACCGTCCGGGCGCGCTTCGTGGCCTCCCGGCTGATGCTCAAGCACACCGCCGGCGCGGTCCTCGGCGAGCCGCCGCACGCCCTGGAACTGGCCTACAAGCTGGGCGGCCGCCCGTACCTGCGCGGTTTCGAACAGCTGGACGTCAGCCTCAGCCACACCGCCGACCTGCTGGTGGTCGGGCTGACCAGGCGCGGCTGGATCGGCGTGGACGCCGAACTCCGTGACCGGCAGATGGTGGGCCTGGGCACCGAGGCCCAGATGTGCACCCGGCACGAGCGGGAGGAGCTGGAGCGGATCGCCGAGGAGCAGCGCAACACCGCGCTGGTGCGCCTGTGGACACTCAAGGAGGCGTACAGCAAGGCCATCGGCCAGGGCATGCGGTTCCGCTTCACCGAGTTCGGCTTCGGGCCGCGGGAGCTCCAGGCGGCGGACGTCCGACTGCCCGACGGCTCCCCCGCGACCGGCGCGGAGTGGACCTTCCGCACCGCCCTGCTGCAGGAGCGCTACACGGTGAGCGTCGCCCTGTACGACGCGGGCTTCGGGGAGGCGGGTGACGGGGCGCACGCGCTGCGGGTGGAGGACGGCCTGCTGGCCGAGCTGCTCGGTGGGGCCGCCGACGCCCCTTGA (1) Construction of recombinant vector containing PPTase B3D0 gene The gene encoding PPTase B3D0 provided by the present invention was synthesized by Qingke Biotechnology Co., Ltd., and pSET28a was used as a vector to construct a PPTase B3D0 protein expression vector pSET28a-B3D0 with a His tag. B3D0 The gene was overexpressed and the promoter was selected as the constitutive promoter PermE*. The plasmid pSET152-PermE* was first double-digested with two restriction endonucleases, 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 ).

[0035] (2) Contains PPTase B3D0 Construction of gene recombinant bacteria B3D0 Construction of gene overexpression recombinant bacteria: The recombinant plasmid pSET152-PermE*-B3D0 was transformed into Escherichia coli ET12567 (pUZ8002), and then the plasmid was introduced into Streptomyces coelicolor M145, Streptomyces icebergensis BC-101-4, Streptomyces avermitilis S0, Saccharopolyspora spinosa NRRL 18395, Streptomyces fissureus M4018, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ISP5230 and Streptomyces albus IPPDNR through inter-genus conjugative transfer experiments, and 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).

[0036] Construction of control recombinant bacteria containing empty vector: By transformation, the empty vector pSET152 was introduced into Escherichia coli ET12567 / pUZ8002. Then, pSET152 was introduced into Streptomyces coelicolor M145, Streptomyces glacialis BC-101-4, Streptomyces avermitilis S0, Saccharopolyspora spinosa NRRL 18395, Streptomyces crassa M4018, Streptomyces roseospora NRRL 11379, Streptomyces venezuelae ISP5230 and Streptomyces albus IPPDNR respectively by the same inter-genus conjugative transfer method to obtain control strains Streptomyces coelicolor M145-C, Streptomyces glacialis BC-101-4-C, Streptomyces avermitilis S0-C, Saccharopolyspora spinosa NRRL 18395-C, Streptomyces crassa M4018-C, Streptomyces roseospora NRRL 11379-C, Streptomyces venezuelae ISP5230-C and Streptomyces albus IPPDNR-C.

[0037] Example 2: Verification of the in vitro catalytic effect of PPTase B3D0 (1) Protein expression and purification The plasmid pSET28a-B3D0 in Example 1 was transferred 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.

[0038] SEQ ID NO.5: GVEAAKVLEDVAGADAPGHGIAEQEHFVTSGFDSAAAVALRNRLNDATGLLLPFTLAFDHPTPAAVADHLHSRL The corresponding ACP gene was amplified from the gene cluster of Streptomyces BC-101-4 using primers ACP-F / R, and the protein expression plasmid was constructed and transferred into BL21. After inducing expression, the purified protein ACP was obtained by purification.

[0039] The specific steps of protein induction expression and purification are as follows: ① Transform the constructed recombinant protein expression plasmid into E. coli BL21 and culture overnight at 37°C and 250 rpm; 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; 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; ④Add appropriate amount of IPTG for induction, culture overnight at 16°C and 200 rpm; ⑤ Centrifuge at 4000 rpm for 10 min to collect the bacteria; ⑥ Resuspend the cells with an appropriate amount of Binding Buffer and then disrupt them with ultrasound; ⑦ Centrifuge at 6000 rpm, 4°C for 30 min to obtain the supernatant and precipitate. Take a small amount of the supernatant and precipitate, add 5× Loading Buffer to resuspend, boil the sample in boiling water for 10 min, and run SDS-PAGE to detect protein expression; ⑧Resuspend the Ni column with Binding Buffer, pass the supernatant through the column, and 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 effluent; take a small amount of the eluate and add 5× Loading Buffer to resuspend, boil the sample in boiling water for 10 min, and run SDS-PAGE to detect the protein expression level.

[0040] The SDS-PAGE gel results 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.

[0041] (2) In vitro reaction and HPLC detection The purified and concentrated target protein was dialyzed in dialysate overnight, and the dialyzed ACP and PPTase B3D0 were added to the prepared lysate 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 carried out at 25°C for 30 min and then quenched with dry ice. Using unreacted ACP as a control, the molecular weight change of ACP was detected by HPLC and SDS-PAGE to determine whether the reaction was proceeding.

[0042] 50 μL of purified ACP and the reaction solution after in vitro reaction were taken for HPLC detection. Protein detection used a C18 reverse phase column; the mobile phase used two phases: 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 min, and the concentration of phase B was 0-75% in the first 15 min, and 75%-100% in 15-20 min.

[0043] The results of HPLC detection of the prosthetic group status of ACP are as follows Figure 2As shown in c in the figure, it can be seen that B3D0 can achieve partial cofactorization of ACP.

[0044] Example 3: Application of PPTase B3D0 in increasing the yield of polyketide milbemycin type I of Streptomyces bingchengensis The recombinant strains BC-101-4-B3D0, BC-101-4-C and the starting strain BC-101-4 of Streptomyces bingchengensis obtained in Example 1 were inoculated on SKYM solid medium, cultured at 28°C for 9 days, and spores of about 1 square centimeter were scraped and inoculated in the seed medium of Streptomyces bingchengensis, and cultured at 28°C, 250 rpm for 46 h. Then, the inoculation amount of 6% was inoculated in the fermentation medium of Streptomyces bingchengensis, and cultured at 28°C, 250 rpm for 9 days.

[0045] The spore-forming medium was SKYM medium, and its specific composition was as follows: 4 g / L sucrose, 1 g / L skimmed milk powder, 2 g / L yeast extract powder, 5 g / L malt extract powder, 20 g / L agar powder, and the balance was water.

[0046] The composition of the seed culture medium: 5 g / L yeast extract powder, 10 g / L sucrose, 1 g / L skimmed milk powder, 3.5 g / L bacterial peptone, 0.5 g / L potassium dihydrogen phosphate, and the balance is water.

[0047] The composition of the fermentation medium: 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 dipotassium hydrogen phosphate, 3 g / L calcium carbonate, and the balance is water.

[0048] Milbemycin detection method: 0.5 mL of the fermentation broth of Streptomyces bingchengensis was collected and mixed with 1.5 mL of ethanol. The treated sample was flipped and shaken 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 phase detection. The detection instrument was 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: within 0-15 minutes, the mobile phase A (methanol) was eluted from 0% to 100%, and the mobile phase B (volume ratio of acetonitrile: methanol: water = 7:2:1) was gradient eluted from 100% to 0%; 15-17 minutes, the 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.

[0049] 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 the yield of milbemycin by 43.41% to 1398.63 mg / L. This indicates that the excavated PPTase element B3D0 is an effective biosynthetic element that can increase the yield of type I polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0050] Example 4: Application of PPTase B3D0 in increasing the yield of polyketide avermectins of Streptomyces avermitilis type I The recombinant strains S0-B3D0, S0-C and the starting strain S0 of Streptomyces avermitilis obtained in Example 1 were inoculated on YMS solid medium and cultured at 28°C for 6 days. A bacterial sheet of about 1 square centimeter was taken by inoculation shovel and inoculated in the seed medium of Streptomyces avermitilis, and cultured at 28°C, 250 rpm for 40 h. Then, an inoculum amount of 6% was inoculated in the fermentation medium of Streptomyces avermitilis, and cultured at 28°C, 250 rpm for 10 days.

[0051] The spore-forming medium was YMS medium, and its specific composition was as follows: 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.

[0052] The composition of the seed culture medium: 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.

[0053] The composition of the fermentation medium: 120 g / L corn starch, 38 g / L soybean cake powder, 0.8 g / L calcium carbonate, 8 g / L yeast extract powder, 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 is water.

[0054] Abamectin detection method: Take 0.25 mL of the fermentation broth or fermentation broth supernatant of Streptomyces avermitilis and mix it thoroughly with 1.20 mL of methanol, and treat the sample ultrasonically for 40 min to extract the total avermectin in the fermentation broth and the extracellular avermectin. Then the sample after preliminary treatment was centrifuged at 12000 rpm for 10 min, and the upper layer of avermectin extract was aspirated and passed through a 0.22 μm organic filter membrane for liquid phase detection. The detection instrument was Shimadzu HPLC (Shimadzu LC), the chromatographic column was 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.

[0055] 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 the yield of avermectin by 28.17% to 2.73 g / L. This indicates that the discovered PPTase element B3D0 is an effective biosynthetic element that can increase the yield of type I polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0056] Example 5: Application of PPTase B3D0 in increasing the yield of polyketide spinosad from Saccharopolyspora spinosa type I The recombinant strains of Saccharopolyspora spinosa NRRL 18395-B3D0, NRRL 18395-C and the starting strain NRRL 18395 obtained in Example 1 were inoculated on YD solid medium and cultured at 28°C for 6 days. Spores were collected with water to prepare a spore suspension, and the corresponding volume of the spore suspension was inoculated into 50 mL seed medium at a 1% inoculation rate, and cultured at 28°C, 250 rpm for 3 days. At a 2% inoculation rate, the seed culture solution in logarithmic phase growth was transferred to the fermentation medium, and cultured at 28°C, 250 rpm for 10 days.

[0057] The spore-forming medium was YD medium, and its specific composition was as follows: 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.

[0058] The composition of the seed medium was: 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.

[0059] The composition of the fermentation medium: 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 is water.

[0060] Spinosad detection method: 5 mL of fermentation broth was mixed with an equal volume of methanol and ultrasonically treated for 30 min to extract total spinosad in the fermentation broth and extracellular spinosad. Centrifuge at 10,000 rpm for 10 min, filter the supernatant through a 0.22 μm microporous filter membrane and perform liquid phase detection. The detection instrument was Shimadzu HPLC (Shimadzu LC), the chromatographic column was reversed phase C18, the volume ratio of the mobile phase was methanol: acetonitrile: water = 40:50:10, the flow rate was 0.5 mL / min, the column temperature was 25°C, the injection volume was 10 μL, and the detection wavelength was 246 nm.

[0061] 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 the production of spinosad by 30.21% to 35.42 mg / L. This indicates that the discovered PPTase element B3D0 is an effective biosynthetic element that can increase the production of type I polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0062] Example 6: Application of PPTase B3D0 in increasing the yield of polyketide actinomycetin type II of Streptomyces coelicolor The recombinant strains of Streptomyces coelicolor obtained in Example 1, M145-B3D0, M145-C, and the original strain M145 were inoculated on MS solid medium, cultured at 28°C for 5 days, and spores were collected. 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.

[0063] The spore-forming medium was MS medium, and its specific composition was as follows: 20 g / L soybean cake powder, 20 g / L mannitol, 20 g / L agar powder, and the balance was water.

[0064] 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 phosphate trihydrate, 25 mM 5×TES buffer, 1 mM sodium dihydrogen phosphate, 1 mM potassium phosphate dihydrate, trace elements: ZnSO4 7H 2 O, NaCl, FeSO 4 7H 2 O,MnCl 2 ·4H 2 O, CaCl 2 6H 2 O each 0.1 g / L, the balance being water.

[0065] 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).

[0066] 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 the production of actinomycete rhodamine by 76.47% to 218.42 mg / L. This indicates that the discovered PPTase element B3D0 is an effective biosynthetic element that can increase the production of type II polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0067] Example 7: Application of PPTase B3D0 in increasing the yield of polyketide oxytetracycline type II of Streptomyces pyrifera The recombinant strains M4018-B3D0 and M4018-C of Streptomyces pyogenes obtained in Example 1 and the original strain M4018 were inoculated on MS solid culture medium, respectively, and cultured at 30°C for 5 days. Spores were scraped and inoculated into Streptomyces pyogenes seed culture medium, and cultured at 30°C and 250 rpm for 24 h. Then, the culture medium was transferred to Streptomyces pyogenes fermentation medium with an inoculum size of 5%, and cultured at 28°C and 250 rpm for 8 days.

[0068] The spore-forming medium was MS medium, and its specific composition was as follows: 20 g / L soybean cake powder, 20 g / L mannitol, 20 g / L agar powder, and the balance was water.

[0069] The composition of the seed culture medium: 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.

[0070] The composition of the fermentation medium: 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.

[0071] Detection method of oxytetracycline: take 1.5 mL of fermentation broth, acidify it with 9 mol / L hydrochloric acid to pH 1.5 to 1.7, centrifuge and take the supernatant through a membrane for HPLC detection. Detection method: The detection instrument is Agilent high performance liquid chromatography (HPLC), using SB-C18 (250 mm×4.6 mm, Agilent, America) chromatographic 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.

[0072] The results of oxytetracycline production test are as follows Figure 7 As shown, compared with the starting strain M4018, the overexpression B3D0 The recombinant strain M4018-B3D0 increased the oxytetracycline production by 69.90% to 2.07 g / L. This indicates that the excavated PPTase element B3D0 is an effective biosynthetic element that can increase the production of type II polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0073] Example 8: Application of PPTase B3D0 in increasing the yield of polyketide jedomycin of Streptomyces venezuelae type II The recombinant Venezuelan Streptomyces strains ISP5230-B3D0, ISP5230-C and the original strain ISP5230 obtained in Example 1 were inoculated on MYM solid culture medium, respectively, and cultured at 30°C for 5 days. Spores were scraped and inoculated into seed culture medium, and then cultured with shaking at 28°C for 20 hours, and then transferred to fermentation medium, and continued to be cultured with shaking at 28°C for 48 hours.

[0074] The spore-forming medium was MYM medium, and its specific composition was as follows: 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.

[0075] The seed culture medium was liquid MYM medium.

[0076] The fermentation medium consisted of MSM liquid medium, glucose and phosphate buffer stock solutions, with the final concentration of glucose being 33 mM and the final concentration of phosphate buffer being 50 μM. Each liter of MSM liquid medium (pH 7.5) contained MgSO 4 0.4 g, MOPS 1.9 g, 9 mL saline solution (the saline solution contains 1 g / 100 mL NaCl and 1 g / 100 mL CaCl 2 aqueous solution), 4.5 mL 0.2 g / 100 mL FeSO 4 7H2 O aqueous solution, 4.5 mL of trace element solution and 7.8 g of L-isoleucine. Each liter of trace element solution contains 880 mg ZnSO 4 7H 2 O, 39 mg CuSO 4 ·5H 2 O, 6.1 mg MnSO 4 ·4H 2 O, 5.7 mg H 3 BO 3 and 3.7 mg (NH 4 ) 6 Mo 7 O 24 ·4H 2 O. Each liter of phosphate buffer stock solution (9 mM) contains 10.5 g K 2 HPO 4 and 4.5 g KH 2 PO 4 .

[0077] 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 with a certain volume of methanol or DMSO, and perform HPLC detection. Detection method: The detection instrument is Agilent high performance liquid chromatography (HPLC), using SB-C18 (250 mm×4.6 mm, Agilent, America) chromatographic 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, injection volume 20 μL.

[0078] The results of the jedomycin production test are as follows Figure 8 As shown, compared with the starting strain ISP5230, the overexpression B3D0 The recombinant strain ISP5230-B3D0 increased the yield of jedomycin by 39.04% to 334.99 μg / mL. This indicates that the discovered PPTase element B3D0 is an effective biosynthetic element that can increase the yield of type II polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0079] Example 9: Application of PPTase B3D0 in increasing the yield of polyketide daunorubicin type II of Streptomyces albus The recombinant strains of Streptomyces albus IPPDNR-B3D0, IPPDNR-C and the original strain IPPDNR obtained in Example 1 were inoculated on MS solid culture medium, respectively, and cultured at 30°C for 3 days. Spores of 1 cm×2 cm were scraped and inoculated into a primary seed culture medium (Tryptone Soy Broth TSB), and cultured at 30°C and 220 rpm for 30 hours. The culture was transferred to a secondary seed culture medium at a 5% inoculum size and cultured at 30°C and 220 rpm for 24 h. The culture was then transferred to a fermentation culture medium at a 10% inoculum size and cultured at 30°C and 250 rpm for 10 days.

[0080] The spore-forming medium was MS medium, and its specific composition was as follows: 20 g / L soybean cake powder, 20 g / L mannitol, 20 g / L agar powder, and the balance was water.

[0081] The composition of the secondary seed culture medium: 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.

[0082] The composition of the fermentation medium: 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 is water.

[0083] Daunorubicin detection method: Take 1 mL of fermentation broth, add 9 mL of methanol, let it stand for 12 h and 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 SB-C18 (250 mm×4.6 mm, Agilent, America) chromatographic 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 is 37℃, gradient washing, injection volume is 20 μL.

[0084] The results are as follows Fig. 9 As shown, compared with the starting strain IPPDNR, overexpression B3D0 The recombinant strain IPPDNR-B3D0 increased the production of daunorubicin by 27.27% to 33.73 μg / mL. This indicates that the discovered PPTase element B3D0 is an effective biosynthetic element that can increase the production of type II polyketide, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0085] Example 10: Application of PPTase B3D0 in increasing the yield of polyether natural product Nanchangmycin from Streptomyces bingchengensis The recombinant strains BC-101-4-B3D0, BC-101-4-C and the starting strain BC-101-4 obtained in Example 1 were inoculated on SKYM solid medium, cultured at 28°C for 9 days, and spores of about 1 square centimeter were scraped and inoculated in the seed medium of Streptomyces bingchengensis, cultured at 28°C, 250 rpm, and cultured for 46 hours. Then, the inoculation amount of 6% was inoculated in the fermentation medium of Streptomyces bingchengensis, cultured at 28°C, 250 rpm, and cultured for 9 days.

[0086] The spore-forming medium was SKYM medium, and its specific composition was as follows: 4 g / L sucrose, 1 g / L skimmed milk powder, 2 g / L yeast extract powder, 5 g / L malt extract powder, 20 g / L agar powder, and the balance was water.

[0087] The composition of the seed culture medium: 5 g / L yeast extract powder, 10 g / L sucrose, 1 g / L skimmed milk powder, 3.5 g / L bacterial peptone, 0.5 g / L potassium dihydrogen phosphate, and the balance is water.

[0088] The composition of the fermentation medium: 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 dipotassium hydrogen phosphate, 3 g / L calcium carbonate, and the balance is water.

[0089] Nanchangmycin detection method: 0.5 mL of the fermentation broth of Streptomyces bingchengensis was collected and 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 12000 rpm for 15 minutes. The upper layer of nanchangmycin extract was aspirated and filtered through a 0.22 μm organic filter membrane for liquid phase detection. The detection instrument was 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 min, 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.

[0090] The results of the Nanchangmycin production test are as follows Fig.10 As shown, compared with the starting strain BC-101-4, the overexpression B3D0The recombinant strain BC-101-4-B3D0 increased the yield of nanchangmycin by 45.33% to 1698.29 mg / L. This indicates that the excavated PPTase element B3D0 is an effective biosynthetic element that can increase the yield of polyether compounds, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0091] Example 11: Application of PPTase B3D0 in increasing the yield of non-ribosomal peptide natural product daptomycin The recombinant strains NRRL 11379-B3D0, NRRL 11379-C of Streptomyces roseosporus obtained in Example 1 and the original strain NRRL 11379 were inoculated on DAI solid culture medium, respectively, and cultured at 28°C for 7 days. Spores were scraped and inoculated into the primary seed culture medium of Streptomyces roseosporus, and cultured at 28°C and 250 rpm for 60 h. Thereafter, the secondary seed culture medium was transferred at an inoculum size of 5%, and cultured at 28°C and 250 rpm for 36 h. Then, the fermentation culture medium of Streptomyces roseosporus was transferred at an inoculum size of 5%, and cultured at 28°C and 250 rpm for 10 days. Starting from 48 h, 0.5 mL of 2% (W / V) sterile sodium decanoate solution was added every 12 h.

[0092] The spore-forming medium was DA1 medium, and its specific composition was as follows: 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.

[0093] The composition of the seed culture medium: 5 g / L glucose, 15 g / L dextrin, 5 g / L bacterial peptone, 5 g / L yeast extract powder, 5 g / L peanut cake powder, 0.5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium carbonate, and the balance is water.

[0094] The composition of the fermentation medium: 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 is water.

[0095] Daptomycin detection method: take 1 mL of fermentation broth, centrifuge twice at 13000 rpm, 4℃ 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 SB-C18 (250 mm×4.6 mm, Agilent, America) chromatographic 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.

[0096] The results of daptomycin production test are as follows Fig.11 As shown, compared with the starting strain NRRL 113792, the overexpression B3D0 The recombinant strain NRRL 11379-B3D0 increased the yield of daptomycin by 71.98% to 389.43 mg / L. This indicates that the discovered PPTase element B3D0 is an effective biosynthetic element that can increase the yield of non-ribosomal peptide compounds, which is of great significance for constructing high-yield strains of natural products from actinomycetes and promoting the industrialization of natural products.

[0097] Although the present invention has been disclosed as above in the form of preferred embodiments, it 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. An application of phosphopantetheinyl transferase B3D0 in promoting the synthesis of natural products, characterized in that: The amino acid sequence of the phosphopantetheine transferase 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.

2. Use of a gene encoding phosphopantetheine transferase B3D0 in promoting the synthesis of natural products, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.2; The natural product is a polyketide compound, a polyether compound or a non-ribosomal peptide compound.

3. Use of a recombinant vector containing a gene encoding phosphopantetheinyl transferase B3D0 in promoting the synthesis of natural products, characterized in that: The nucleotide sequence of the gene encoding phosphopantetheine 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.

4. The use according to claim 3, characterized in that: The construction method of the recombinant vector comprises the following steps: constructing a recombinant expression vector pSET28a-B3D0 containing a gene encoding phosphopantetheine transferase B3D0, performing double restriction 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.

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

3.

6. Use of a recombinant bacterium containing the coding gene described in claim 2 or the recombinant vector described in claim 3 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.

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

8. The use according to claim 7, characterized in that: The Escherichia coli is ET12567 (pUZ8002).

9. The use according to claim 7, characterized in that: The starting strain is Streptomyces icebergii ( Streptomyces bingchenggensis )BC-101-4, Streptomyces coelicolor ( Streptomyces coelicolor )M145, Streptomyces avermitilis ( Streptomyces avermitilis )S0, Streptomyces fissipes ( 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.

10. The use according to claim 9, characterized in that: When the starting strain is Streptomyces BC-101-4, the recombinant bacteria is used to increase the production of milbemycin or nanchangmycin; when the starting strain is Streptomyces coelicolor M145, the recombinant bacteria is used to increase the production of actinomycin; when the starting strain is Streptomyces avermitilis S0, the recombinant bacteria is used to increase the production of avermectin; when the starting strain is Streptomyces fissures M4018, the recombinant bacteria is used to increase the production of oxytetracycline; when the starting strain is Saccharopolyspora spinosa NRRL 18395, the recombinant bacteria is used to increase the production of spinosad; when the starting strain is Streptomyces roseosporeus NRRL 11379, the recombinant bacteria is used to increase the production of daptomycin; when the starting strain is Streptomyces venezuelae ISP5230, the recombinant bacteria is used to increase the production of jedomycin; when the starting strain is Streptomyces albus IPPDNR, the recombinant bacteria is used to increase the production of daunorubicin.

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