Application of NADH kinase element sPos5 in improving yield of secondary metabolites of actinomycetes

Abstract

The invention discloses application of an NADH kinase element sPos5 in improving the yield of secondary metabolites of actinomycetes, and belongs to the technical field of genetic engineering. In order to improve the yield of secondary metabolites of actinomycetes, an NADH kinase element sPos5 with a nucleotide sequence as shown in SEQ ID NO.1 is obtained by screening NADH kinase capable of improving intracellular NADPH reducing power of a strain, a recombinant vector and recombinant bacteria are constructed by utilizing the NADH kinase element, and the recombinant bacteria can be used for preparing the secondary metabolites of the actinomycetes. Fermentation experiments show that the NADH kinase element sPos5 can meet the requirement for reducing power in the secondary metabolite synthesis process, and the yield of the secondary metabolite of actinomycetes is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of NADH kinase element sPos5 in improving the yield of secondary metabolites of actinomycetes. Background Art

[0002] Actinomycetes are important strains in the industrial production of natural product pesticides. For example, avermectin, the biopesticide with the largest global production and usage, and spinosad, jinggangmycin, and milbemycin, which are widely used in the prevention and control of various crop pests and diseases, are all produced by actinomycete fermentation. However, these natural product pesticides are secondary metabolites that are not essential for the growth of actinomycetes. They all face common problems such as low product yield and difficulty in strain optimization during the fermentation process, which has become a research bottleneck restricting the creation and promotion of natural product pesticides.

[0003] From a certain perspective, there is a great degree of competition between the secondary metabolism and primary metabolism of the strain. In this process, the secondary metabolic pathway is naturally at a disadvantage, which is also one of the important reasons for the low yield of secondary metabolites. Metabolic competition includes carbon source competition and cofactor competition, among which cofactor competition is often easily overlooked. At present, there are few studies on the modification of the cofactor pathway of the secondary metabolism of actinomycetes. However, the synthesis of secondary metabolites of actinomycetes usually has a large demand for energy. For example, 11, 14 and 5 molecules of NADPH are required for each molecule of avermectin, milbemycin and daunorubicin, respectively. NADPH is mainly produced by the pentose phosphate pathway. However, in the synthesis stage of secondary metabolites, the activity of the pentose phosphate pathway is significantly reduced, which leads to a significant decrease in the intracellular NADPH concentration, which gradually becomes an important factor affecting the biosynthesis of secondary metabolites, especially polyketides and non-ribosomal peptides. In the secondary metabolism stage, NADH is relatively sufficient due to the large-scale degradation of intracellular neutral lipids. Since NADH kinase is an important enzyme that catalyzes NADH to generate NADPH, screening for efficient NADH kinase to increase the intracellular NADPH concentration of the strain is of great significance for increasing the production of compounds such as polyketides, polyethers and non-ribosomal peptides in actinomycetes. Summary of the invention

[0004] In order to increase the yield of secondary metabolites of actinomycetes, the present invention screened NADH kinases that can improve the reducing power of intracellular NADPH in the strain, obtained an NADH kinase element sPos5 with a nucleotide sequence as shown in SEQ ID NO.1, constructed a recombinant vector and recombinant bacteria using the NADH kinase element, and found through fermentation experiments that the NADH kinase element sPos5 can meet the demand for reducing power in the synthesis process of secondary metabolites, thereby increasing the yield of secondary metabolites of actinomycetes.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide an application of a NADH kinase element sPos5 in increasing the yield of secondary metabolites of actinomycetes, wherein the nucleotide sequence of the NADH kinase element sPos5 is shown in SEQ ID NO.1, and the secondary metabolites are polyketide compounds, polyether compounds or non-ribosomal peptide compounds.

[0007] The second object of the present invention is to provide a recombinant vector containing the above-mentioned NADH kinase element sPos5 for use in increasing the yield of secondary metabolites of actinomycetes, wherein the secondary metabolites are polyketide compounds, polyether compounds or non-ribosomal peptide compounds.

[0008] In one embodiment of the present invention, the starting vector of the recombinant vector is pSET152, and the promoter actⅡ-orf4p fragment is amplified using the genome of Streptomyces coelicolor as a template, and the promoter actⅡ-orf4p fragment and the NADH kinase element sPos5 fragment are integrated into the starting vector to obtain the recombinant vector pSET152::orf4 sPos5.

[0009] In one embodiment of the present invention, the nucleotide sequences of the upstream primer and the downstream primer used to amplify the promoter actⅡ-orf4p fragment are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0010] In one embodiment of the present invention, the recombinant vector pSET152::orf4 sPos5 is used to increase the yield of actinomycetophthrin produced by fermentation of Streptomyces coelicolor.

[0011] In one embodiment of the present invention, the starting vector of the recombinant vector is pSET152::ermEp*, and the NADH kinase element sPos5 fragment is integrated into the starting vector to obtain the recombinant vector pSET152::ermEp*sPos5.

[0012] In one embodiment of the present invention, the recombinant vector is used to increase the production of milbemycin or nanchangmycin in Streptomyces bingchenggensis, to increase the production of avermectin in Streptomyces avermitilis, to increase the production of FK506 in Streptomyces tsukubaensis, to increase the production of oxytetracycline in Streptomyces rimosus, to increase the production of daptomycin in Streptomyces roseosporus, to increase the production of daptomycin in Streptomyces venezuelae, to increase the production of daunorubicin in Streptomyces albus, or to increase the production of spinosad in Saccharopolyspora spinosa.

[0013] The third object of the present invention is to provide a recombinant bacterium containing the above-mentioned recombinant vector pSET152::orf4 sPos5 for use in increasing the yield of actinomycetophthrin produced by fermentation of Streptomyces coelicolor. The recombinant bacterium is obtained by taking Streptomyces coelicolor M145 as a starting strain and introducing the recombinant vector pSET152::orf4 sPos5 into the starting strain.

[0014] The fourth object of the present invention is to provide a recombinant bacterium containing the above-mentioned recombinant vector pSET152::ermEp*sPos5 for improving the yield of secondary metabolites of actinomycetes, wherein the recombinant bacterium is obtained by introducing the recombinant vector pSET152::ermEp*sPos5 into the starting strain, using Streptomyces bingchengensis BC-101-4, Streptomyces avermitilis S0, Streptomyces tsukuba NRRL 18488, Streptomyces crassifolius M4018, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ISP5230, Streptomyces albus IPPDNR or Saccharopolyspora spinosa NRRL 18395 as the starting strain.

[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 avermitilis S0, the recombinant bacteria is used to increase the yield of avermectin; when the starting strain is Streptomyces tsukuba NRRL 18488, the recombinant bacteria is used to increase the yield of FK506; when the starting strain is Streptomyces crassa M4018, the recombinant bacteria is used to increase the yield of oxytetracycline; when the starting strain is Streptomyces roseospores 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; when the starting strain is Saccharopolyspora spinosa NRRL 18395, the recombinant bacteria is used to increase the yield of spinosad.

[0016] Beneficial effects of the present invention:

[0017] The present invention obtains an NADH kinase element sPos5 with a nucleotide sequence as shown in SEQ ID NO.1 by screening NADH kinases capable of improving the reducing power of intracellular NADPH of a strain, and constructs a recombinant vector and a recombinant bacterium using the NADH kinase element. Fermentation experiments show that the NADH kinase element sPos5 can meet the demand for reducing power in the synthesis of secondary metabolites, thereby improving the yield of secondary metabolites of actinomycetes.

[0018] The invention constructs a recombinant vector and a recombinant strain by mining efficient NADH kinase, and obtains 9 high-yield recombinant strains overexpressing NADH kinase element sPos5, namely, Streptomyces coelicolor M145 / CN, Streptomyces bingchengensis BC-101-4 / CN, Streptomyces avermitilis S0 / CN, Streptomyces tsukuba NRRL 18488 / CN, Streptomyces crassifolius M4018 / CN, Streptomyces roseosporus NRRL 11379 / CN, Streptomyces venezuelae ISP5230 / CN, Streptomyces albus IPPDNR / CN and Saccharopolyspora spinosa NRRL18395 / CN. Among them, the actinomycin production of the recombinant strain Streptomyces coelicolor M145 / CN was increased by 76.02% compared with the starting strain M145, reaching 161.12 mg / L; the milbemycin production of the recombinant strain Streptomyces icytogenes BC-101-4 / CN was increased by 32.25% compared with the starting strain BC-101-4, reaching 1285.97 mg / L, and the production of nanchangmycin was increased by 36.47%, reaching 1530.58 mg / L; the avermectin B of the recombinant strain Streptomyces avermitilis S0 / CN was increased by 14.8% compared with the starting strain BC-101-4. 1aThe yield was increased by 33.16% compared with the starting strain S0, reaching 2.99 g / L; the FK506 yield of the recombinant strain Streptomyces tsukuba NRRL 18488 / CN was increased by 91.96% compared with the starting strain NRRL 18488, reaching 110.37 mg / L; the oxytetracycline yield of the recombinant strain Streptomyces crisscrossing M4018 / CN was increased by 56.43% compared with the starting strain M4018, reaching 2.04 g / L; the daptomycin yield of the recombinant strain Streptomyces roseosporus NRRL 11379 / CN was increased by 56.43% compared with the starting strain NRRL 11379 increased by 35.06% to 329.57 mg / L; the jendomycin production of the recombinant strain Venezuelan Streptomyces ISP5230 / CN increased by 23.43% compared with the starting strain ISP5230, reaching 308.42 μg / mL; the daunorubicin production of the recombinant strain white Streptomyces IPPDNR / CN increased by 30.52% compared with the starting strain IPPDNR, reaching 31.82 μg / mL; the spinosad production of the recombinant strain Saccharopolysaccharides NRRL18395 / CN increased by 30.67% compared with the starting strain NRRL 18395, reaching 37.64 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the construction of the recombinant vector pSET152::orf4 sPos5;

[0020] Figure 2 Schematic diagram of the construction of the recombinant vector pSET152::ermEp*sPos5;

[0021] Figure 3 The graph is a test result of the actinomycetoma production of Streptomyces coelicolor recombinant strains M145 / CN, M145 / C and the starting strain M145;

[0022] Figure 4 The figure shows the test results of the production of milbemycin and nanchangmycin of the recombinant strains BC-101-4 / CN, BC-101-4 / C of Streptomyces bingchengensis and the starting strain BC-101-4; among which, Figure 4 A in the figure is the test result of milbemycin production. Figure 4 B in the figure is the test result diagram of Nanchangmycin yield;

[0023] Figure 5 The recombinant strains S0 / CN, S0 / C and the original strain S0 avermectin B 1a Output test result chart;

[0024] Figure 6The figure is the test result of the yield of the recombinant strains of Streptomyces tsukubaensis NRRL 18488 / CN, NRRL 18488 / C and the starting strain NRRL18488FK506;

[0025] Figure 7 The graph is a test result of oxytetracycline production of the recombinant strains M4018 / CN, M4018 / C of Streptomyces fissiliflorus and the starting strain M4018;

[0026] Figure 8 The figure is the test result of daptomycin production of Streptomyces roseosporus recombinant strains NRRL 11379 / CN, NRRL 11379 / C and the starting strain NRRL11379;

[0027] Fig. 9 The graph is the test result of the yield of jedomycin of the recombinant strains ISP5230 / CN, ISP5230 / C and the starting strain ISP5230 of Streptomyces venezuelae;

[0028] Fig.10 It is the test result diagram of daunorubicin production of Streptomyces albus recombinant strains IPPDNR / CN, IPPDNR / C and starting strain IPPDNR;

[0029] Fig.11 This is a graph showing the test results of the spinosad production of the recombinant Saccharopolyspora strains NRRL 18395 / CN, NRRL 18395 / C and the starting strain NRRL18395. DETAILED DESCRIPTION

[0030] 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.

[0031] 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.

[0032] Streptomyces coelicolor M145 is disclosed in the following document: Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Foundation, Norwich.

[0033] Streptomyces bingchenggensis BC-101-4 is disclosed in the following document: Wang X, Wang X, Xiang W (2009) Improvement of milbemycin-producing Streptomyces bingchenggensis by rational screening of ultraviolet-and chemically induced mutants. World J Microbiol Biotechnol 25: 1051-1056.

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

[0035] Streptomyces tsukubaensis NRRL 18488 is disclosed in the following literature: Ma Dongxu. Improving tacrolimus production by regulating the expression of proteins encoded by BulZ and its target genes [D]. Tianjin: Tianjin University, 2018. DOI: 10.7666 / d.D01679402.

[0036] Streptomyces rimosus M4018 is disclosed in the following literature: Yin Shouliang, Lin Zhiwei, Zhang Yuxiu, et al. Engineering Streptomyces rimosus to improve oxytetracycline production [J]. Chinese Journal of Biotechnology, 2016, 36(7): 72-82. DOI: 10.13523 / j.cb.20160711.

[0037] Streptomyces roseosporus NRRL 11379 is disclosed in the following literature: Yu Jisan, Hong Kui, Lin Haipeng, et al. Optimization of fermentation medium for production of daptomycin precursor A21978C by Streptomyces roseosporus NRRL11379 [J]. Anhui Agricultural Sciences, 2008, 36(19): 7974-7976. DOI: 10.3969 / j.issn.0517-6611.2008.19.012.

[0038] 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. DOI: 10.3969 / j.issn.1006-2858.2006.11.013.

[0039] IPPDNR of Streptomyces albus was constructed by using Streptomyces albus Del14 as the starting strain and heterologously expressing the synthetic gene cluster of daunorubicin. Streptomyces albus Del14 was disclosed in the following documents: MYRONOVSKYI M, B, NADMID S, et al. Generation of cluster-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 dnrU ketoreductase and dnrV genes and the doxAcytochrome P-450hydroxylase 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.

[0040] Saccharopolyspora spinosa NRRL 18395 is disclosed in the following literature: 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. DOI: 10.3969 / j.issn.1000-2421.2012.03.007.

[0041] The above strains were stored in our laboratory.

[0042] The strain information involved in the following examples is shown in Table 1, the primer information involved is shown in Table 2, and the plasmid information involved is shown in Table 3.

[0043] Table 1 Strain information involved in the examples

[0044]

[0045]

[0046] Table 2 Primer information involved in the examples

[0047]

[0048] Note: Bold characters represent homology arm sequences; underlined characters represent restriction endonuclease sites.

[0049] Table 3 Plasmid information involved in the examples

[0050]

[0051] Example 1: Construction of a recombinant vector containing the NADH kinase element sPos5

[0052] In order to obtain an efficient NADH kinase element that improves the intracellular NADPH reducing power, the inventors analyzed all the 268 Streptomyces strains in the database that were annotated as NAD + / NADH kinase elements, a total of 349 sequences were obtained. Among these sequences, a NADH kinase element sPos5 from Streptomyces albus was obtained through homology comparison and analysis. The nucleotide sequence of sPos5 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0053] SEQ ID NO.1:

[0054] ATGGTGACTGAACCCGGATGGGCCGCCCGCGCCCTGCTCATCGCCAACCCCCAGGCCGGGGTCCGCGACCGGCAGCACATCGACGAGGTCATCGCCCACTGCCGAGGCCTGGTGCCGGGGCTCGACGTCGTGCACACCGAGTACCAGGGCCACGCCGAGGCGAGCGCCGCCGAGGCCGCCGCCGCGGGGTACGACGCGGTGATCGTGCTCGGCGGTGACGGCACGACCCGCGAGGCGGCCTCGGGCCTGGCCCGCGCGGGCCTCGAACAGCCCGCGGGCAAGCGCCCCGCGATGGTCAACATCCCCTTCGGCACCGGGAATTCGTTCTACCA GGAGATCTGGGCCGACGCCCCCTGGAGCGCGGTCCTGGACCAGGCGCTCTCCGGCGAGCAGCCGCATCTGCGCTGGGTGGACATGGCGCACATCCGGGAGATCGACGTCCTGGCGCTGCTCGGCGCGGGCTCGGGCCTGGTCGCCGACGCCTTGGAGGCGGCCTACGGCATGTTCGAGGTGCCCGGCCGCGACCGCTACCAGCAGGCCGTCGCCCAGACCATGGCCACCTTCACGCCGTACGAGGGCCGGGTGAGCGTGGACGGCAGGGTGGTCCACGAGGGCCCCGTCGTCCTGGTGAACATCGGCGGCGGCCGCTACCGGGCGGGCCGGTTCAAGCTGCTGCCGCACTCGGTCATCGACGACGGACTGCTCGACGTGTGCGTGGTGGGCGGCCAGATGGGCGTACGCGAGCTGGCCGGGCTGACCGCGGACGGCAGCCACATCGGGCGCCCCGGCGTGGTGTACGAGCGCGGCTCCCGCTTCGTCGTGGAGCGCACCGACGGCAGGAAGCTCTCCTTCGAGCACGACGGCGAACTCTGCACGGGCGAGGCCTCGCGCTACACCATCGACGTGCTCCCCGCCGTCCTGCCCGTCCTGGCCCCGCCGGCGGCGGTCGCGCACGCCTCCGGCTCCGCGGAGCTCGCCGAGGGCGTCGCATGA

[0055] SEQ ID NO.2:

[0056] MVTEPGWAARALLIANPQAGVRDRQHIDEVIAHCRGLVPGLDVVHTEYQGHAEASAAEAAAAGYDAVIVLGGDGTTREAASGLARAGLEQPAGKRPAMVNIPFGTGNSFYQEIWADAPWSAVLDQALSGEQPHLRWVDMAHIREIDVLALLGAGSGLVADALEAA YGMFEVPGRDRYQQAVAQTMATFTPYEGRVSVDGRVVHEGPVVLVNIGGGRYRAGRFKLLPHSVIDDGLLDVCVVGGQMGVRELAGLTADGSHIGRPGVVYERGSRFVVERTDGRKLSFEHDGELCTGEASRYTIDVLPAVLPVLAPPAAVAHASGSAELAEGVA

[0057] (I) Construction of a recombinant vector expressing the NADH kinase element sPos5 in Streptomyces coelicolor:

[0058] In Streptomyces coelicolor, the efficient NADH kinase element sPos5 was expressed using the sequential promoter actⅡ-orf4p. The specific method for constructing the recombinant vector was as follows: primers 4-sPos5-F and sPos5-R were designed using the whole genome sequence of Streptomyces albus as a template, and the sPos5 fragment was obtained by PCR using the KOD high-fidelity enzyme system; primers Orf4-F and Orf4-R were designed using the genome of Streptomyces coelicolor as a template, and a promoter (actⅡ-orf4p) fragment with homology arms was obtained by PCR using the KOD high-fidelity enzyme system.

[0059] The plasmid pSET152 was double-digested with EcoRI and XbaI to obtain a linear vector xSET152, and the linear vector xSET152 was assembled with the fragments actⅡ-orf4p and sPos5 with homology arms by Gibson. After the reaction, the transformation was completed, a single clone was picked for culture, and the plasmid was extracted with a plasmid extraction kit. After electrophoresis detection, sequencing verification was performed, and the correct recombinant plasmid vector pSET152::orf4 sPos5 was obtained after verification. The schematic diagram of the construction of the recombinant plasmid vector is shown in the figure. Figure 1 shown.

[0060] (II) Construction of recombinant vectors expressing NADH kinase element sPos5 in Streptomyces bingchengensis, Streptomyces avermitilis, Streptomyces tsukuba, Streptomyces guillotineus, Streptomyces roseosporus, Streptomyces venezuelae, Streptomyces albus and Saccharopolyspora spinosa:

[0061] In Streptomyces bingchengensis BC-101-4, Streptomyces avermitilis S0, Streptomyces tsukubaensis NRRL 18488, Streptomyces crassifolius M4018, Streptomyces roseosporus NRRL 11379, Venezuelan Streptomyces ISP5230, white Streptomyces IPPDNR and spiny polycysta NRRL18395, the high-efficiency NADH kinase element sPos5 was expressed by the constitutive strong promoter ermEp*, and the specific construction method of the recombinant vector was as follows: the whole genome sequence of white Streptomyces was used as a template to design primers E-sPos5-F and sPos5-R, and the sPos5 fragment was obtained by PCR using the KOD high-fidelity enzyme system; the plasmid pSET152::ermEp* was double-digested with two restriction endonucleases KpnI and BamHI to obtain a linear vector xSET152-2, and the linear vector xSET152-2 and the fragment sPos5 with homology arms were Gibson assembled, and after the reaction was completed, the transformation was carried out, a single clone was picked for culture, and the plasmid was extracted with a plasmid extraction kit, and the electrophoresis detection was followed by sequencing verification. After verification, the correct recombinant plasmid vector pSET152::ermEp*sPos5 was obtained. The construction schematic diagram of the recombinant plasmid vector is shown in Figure 2 shown.

[0062] Example 2: Construction of recombinant bacteria containing NADH kinase element sPos5

[0063] (I) Construction of a recombinant strain overexpressing the NADH kinase element sPos5

[0064] The recombinant plasmid vector pSET152::orf4 sPos5 obtained in Example 1 was transformed into the competent Escherichia coli ET12567 (pUZ8002), and then the plasmid was introduced into Streptomyces coelicolor M145 through an inter-genus conjugative transfer experiment (see the following literature: Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. The John Innes Foundation, Norwich.). After the conjugates grew, they were picked on MS medium containing apramycin (Apr) and nalidixic acid (Nal). After resistance verification and PCR verification, the recombinant Streptomyces coelicolor strain M145 / CN overexpressing the NADH kinase element sPos5 was obtained.

[0065] The recombinant plasmid vector pSET152::ermEp*sPos5 obtained in Example 1 was transformed into the competent Escherichia coli ET12567 (pUZ8002), and then the plasmid was introduced into Streptomyces bingchengensis BC-101-4, Streptomyces avermitilis S0, Streptomyces tsukuba NRRL 18488, Streptomyces crassifolius M4018, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ISP5230, Streptomyces albus IPPDNR and Saccharopolyspora spinosa NRRL 18395 respectively by inter-genus conjugative transfer experiment. The inter-genus conjugative transfer experiment of Streptomyces bingchengensis BC-101-4 was referred to the following literature: Zhang Y, He H, Liu H, Wang H, Wang X, Xiang W. Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis. Microb Cell Fact.2016;15(1):152.doi:10.1186 / s12934-016-0552-1;For the intermolecular transfer experiment of Streptomyces avermitilis S0, please refer to the following literature: DONG et al.Applied Microbiology and Biotechnology,2024,108(1).DOI:10.1007 / s00253-023-12964-9;For the intermolecular transfer experiment of Streptomyces tsukubaensis NRRL18488, please refer to the following literature: Martínez-Castro M et al.Appl Microbiol Biotechnol.2013Mar;97(5):2139-52.DOI:10.1007 / s00253-012-4364-x;For the intermolecular transfer experiment of Streptomyces crassifolius M4018, please refer to the following literature: Yin et al.Microb Cell Fact.2015Apr 2;14:46.DOI:10.1186 / s12934-015-0231-7; The inter-generic conjugation experiment of Streptomyces roseosporus NRRL 11379 refers to the following literature: Zhang et al.Appl Environ Microbiol.2015Jun;81(11):3753-65.DOI:10.1128 / AEM.00057-15; The inter-generic conjugation experiment of Streptomyces venezuelae ISP5230 refers to the following literature: Zhang et al.Mol Microbiol.2013Nov;90(4):884-97.DOI:10.1111 / mmi.12406 and Doull et al.J Ind Microbiol.1994Mar;13(2):120-5.DOI:10.1007 / BF01584109. After resistance verification and PCR verification, the recombinant strains of Streptomyces bingchengensis BC-101-4 / CN, Streptomyces avermitilis S0 / CN, Streptomyces tsukuba NRRL 18488 / CN, Streptomyces crassa M4018 / CN, Streptomyces roseosporus NRRL 11379 / CN, Streptomyces venezuelae ISP5230 / CN, Streptomyces albus IPPDNR / CN and Saccharopolyspora spinosa NRRL 18395 / CN overexpressing NADH kinase element sPos5 were obtained. .

[0066] (II) Construction of control recombinant strain containing empty vector:

[0067] The empty vector pSET152 was introduced into Escherichia coli ET12567 / pUZ8002 by transformation. Then, pSET152 was introduced into Streptomyces coelicolor M145, Streptomyces icercifolius BC-101-4, Streptomyces avermitilis S0, Streptomyces tsukuba NRRL 18488, Streptomyces crisscrossing M4018, Streptomyces roseospora NRRL 11379, Streptomyces venezuelae ISP5230, Streptomyces albus IPPDNR and Saccharopolyspora spinosa NRRL 18395 by the same inter-genus conjugative transfer method, and the control strains Streptomyces coelicolor M145 / C, Streptomyces icercifolius BC-101-4 / C, Streptomyces avermitilis S0 / C, Streptomyces tsukuba NRRL 18488 / C, Streptomyces crisscrossing M4018 / C, Streptomyces roseospora NRRL 11379 / C, Streptomyces venezuelae ISP5230 / C, Streptomyces albus IPPDNR / C and Saccharopolyspora spinosa NRRL 18395 / C were obtained.

[0068] Example 3: Application of Streptomyces coelicolor M145 / CN overexpressing NADH kinase element sPos5 in increasing the production of actinomycetophthora indole

[0069] The recombinant strains of Streptomyces coelicolor obtained in Example 2, M145 / CN, M145 / C, and the starting 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 amount of 1000 g / 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.

[0070] 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.

[0071] 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 dihydrogen phosphate trihydrate, 25 mM 5×TES buffer, 1 mM sodium dihydrogen phosphate, 1 mM potassium dihydrogen phosphate, trace elements: ZnSO 4 7H 2 O, NaCl, FeSO 4 7H 2 O,MnCl 2 ·4H 2 O, CaCl 2 6H 2 O 0.1g / L each, and the balance is water.

[0072] 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).

[0073] The test results of actinomycin production are as follows Figure 3 As shown, compared with the starting strain M145, the actinomycin production of the recombinant strain M145 / CN of Streptomyces coelicolor increased by 76.02% to 161.12 mg / L, and the empty control strain M145 / C had no effect on the production of actinomycin.

[0074] Example 4: Application of Streptomyces BC-101-4 / CN overexpressing NADH kinase element sPos5 in increasing the yield of milbemycin and nanchangmycin

[0075] The recombinant strains BC-101-4 / CN, BC-101-4 / C of Streptomyces glacialis obtained in Example 2 and the starting strain BC-101-4 were inoculated on SKYM solid culture medium, cultured at 28°C for 9 days, and spores of about 1 square centimeter were scraped and inoculated in the seed culture medium of Streptomyces glacialis, and cultured at 28°C and 250rpm for 46h. Then, the inoculation amount of 6% was inoculated in the fermentation medium of Streptomyces glacialis, and cultured at 28°C and 250rpm for 9 days.

[0076] The spore-forming medium was SKYM medium, and the 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.

[0077] The composition of the seed culture medium: 5g / L yeast extract powder, 10g / L sucrose, 1g / L skimmed milk powder, 3.5g / L bacterial peptone, 0.5g / L dipotassium hydrogen phosphate, and the balance is water.

[0078] The composition of the fermentation medium: 80g / L sucrose, 20g / L soybean cake powder, 1g / L skimmed milk powder, 0.1g / L ferrous sulfate, 1g / L dipotassium hydrogen phosphate, 3g / L calcium carbonate, and the balance is water.

[0079] Detection method of milbemycin and nanchangmycin: 0.5 mL of the fermentation broth of Streptomyces bingchengensis was collected and mixed with 1.5 mL of ethanol. The treated sample was shaken for 30 minutes to extract the milbemycin in the fermentation broth. The preliminarily treated sample was then centrifuged at 12000 rpm for 15 minutes. The upper milbemycin extract was filtered through a 0.22 μm organic filter membrane for liquid phase detection. The detection instrument was Agilent 1260 high performance liquid chromatography (HPLC), the chromatographic column is a C18 column (Zorbax, 4.6mm×250mm, 5μm), the detection wavelength is 242nm, the injection volume is 20μL, the mobile phase flow rate is 1.0mL / min, and the specific chromatographic conditions are as follows: mobile phase A (methanol) is eluted from 0% to 100% within 0-15min, and mobile phase B (volume ratio of acetonitrile:methanol:water=7:2:1) is gradient eluted from 100% to 0%; 15-17min, mobile phase A is maintained at 100%; 17-25min, mobile phase A (methanol) is eluted from 100% to 0%, and mobile phase B is gradient eluted from 0% to 100%; 25-27min, mobile phase B is maintained at 100%, and the column temperature is 28°C.

[0080] The results of the milbemycin production test are as follows Figure 4 As shown, compared with the starting strain BC-101-4, the milbemycin production of the recombinant strain BC-101-4 / CN of Streptomyces bingchengensis increased by 32.25% to 1285.97 mg / L, and the production of nanchangmycin increased by 36.47% to 1530.58 mg / L, and the empty control strain BC-101-4 / C had no effect on the production of milbemycin and nanchangmycin.

[0081] Example 5: Application of Streptomyces avermitilis S0 / CN overexpressing NADH kinase element sPos5 in improving avermectin production

[0082] The recombinant strains S0 / CN, S0 / C and the starting strain S0 of Streptomyces avermitilis obtained in Example 2 were inoculated on YMS solid medium and cultured at 28°C for 7 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, 250rpm for 48h. Then, the inoculation amount of 6% was inoculated in the fermentation medium of Streptomyces avermitilis, and cultured at 28°C, 250rpm for 10 days.

[0083] 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.

[0084] The composition of the seed culture medium: 25g / L corn starch, 2g / L soybean cake powder, 15g / L peanut cake powder, 4g / L yeast extract powder, 0.026g / L cobalt chloride hexahydrate, 0.03g / L α-amylase, and the balance is water.

[0085] The composition of the fermentation medium: 120g / L corn starch, 38g / L soybean cake powder, 0.8g / L calcium carbonate, 8g / L yeast extract powder, 0.02g / L cobalt chloride hexahydrate, 0.1g / L α-amylase, 0.3g / L ammonium sulfate, 0.022g / L sodium molybdate monohydrate, 0.0023g / L manganese sulfate monohydrate, and the balance is water.

[0086] The detection method of avermectin is as follows: 0.25 mL of the fermentation liquid or the supernatant of the fermentation liquid of Streptomyces avermitilis is mixed with 1.20 mL of methanol, and the sample is ultrasonically treated for 40 min to extract the total avermectin in the fermentation liquid and the extracellular avermectin. Subsequently, the sample after preliminary treatment is centrifuged at 12000 rpm for 10 min, the upper avermectin extract is drawn, and liquid phase detection is performed after passing through a 0.22 μm organic filter membrane. The detection instrument is Shimadzu HPLC (Shimadzu LC), the chromatographic column is a C18 column (Zorbax, 4.6 mm×250 mm, 5 μm), the detection wavelength is 246 nm, the injection volume is 20 μL, the mobile phase is 90% methanol, the flow rate is 1.0 mL / min, and the column temperature is 35° C.

[0087] Avermectin B 1a The test results of yield are as follows Figure 5 As shown, compared with the starting strain S0, the avermectin B of the recombinant strain S0 / CN of Streptomyces avermitilis 1a The yield increased by 33.16% to 2.99 g / L, and the empty control strain S0 / C had a significant effect on avermectin B. 1a The production volume has no impact.

[0088] Example 6: Application of Streptomyces tsukubaensis NRRL 18488 / CN overexpressing NADH kinase element sPos5 in increasing FK506 yield

[0089] The recombinant strains of Streptomyces tsukubaensis NRRL 18488 / CN, NRRL 18488 / C and the starting strain NRRL 18488 obtained in Example 2 were inoculated on spore-forming solid culture media, respectively, and cultured at 28°C for 14 days. Spores were scraped and inoculated into Streptomyces tsukubaensis YEME seed culture media, cultured at 28°C and 220rpm for 48h, and then transferred to MGm fermentation medium at an inoculum size of 6%, and cultured at 28°C and 220rpm for 10 days.

[0090] The composition of the spore-forming solid medium is as follows: 10 g / L soluble starch, 1 g / L NaCl, 2 g / L (NH 4 ) 2 SO 4 , 1g / LK 2 HPO 4 , 2g / L CaCO 3 , 1g / L MgSO 4 7H 2 O, 0.001 g / L FeSO 4 7H 2 O, 0.001 g / L MnCl 2 ·4H 2 O, 0.001g / L ZnSO 4 7H 2 O, 20 g / L agar, the balance is water, pH 7.0.

[0091] The composition of YEME seed culture medium is as follows: 3 g / L yeast extract powder, 5 g / L peptone, 3 g / L malt extract powder, 10 g / L glucose, 340 g / L sucrose, and the balance is water, pH 7.0.

[0092] The composition of the MGm fermentation medium was as follows: 50 g / L soluble starch, 8.83 g / L glutamic acid, 2.5 mM K 2 HPO 4 , 0.2 g / L MgSO 4 7H 2 O, 1 mg / L CaCl 2 , 1mg / LNaCl, 0.009g / L FeSO 4 7H 2 O, 21g / L MOPS, 0.45ml / L trace elements. Each 10ml trace elements contains 39.0mg CuSO 4 ·5H 2 O, 5.7 mg H3 PO 3 , 3.7mg(NH 4 ) 6 MO 7 O 24 ·4H 2 O, 6.1 mg MnSO 4 ·H 2 O, 880.0 mg ZnSO 4 7H 2 O, balance water, pH 6.5.

[0093] FK506 detection method: Take 0.5mL fermentation broth in 0.75mL methanol, place in a 50℃ constant temperature water bath for 2.5 hours, shake once every 30 minutes, centrifuge and take the supernatant through a membrane for HPLC detection. Detection method: Use SB-C18 (250mm×4.6mm, Agilent, America) chromatographic column, the volume ratio of the mobile phase is acetonitrile: 0.1% phosphoric acid solution = 65:35, the flow rate is 1.0mL / min, the column temperature is 50℃, the detection wavelength is 210nm, and the injection volume is 20μL.

[0094] The test results of FK506 production are as follows Figure 6 As shown, compared with the starting strain NRRL 18488, the FK506 yield of the recombinant strain NRRL 18488 / CN of Streptomyces tsukubaensis increased by 91.96% to 110.37 mg / L, and the empty control strain NRRL18488 / C had no effect on the yield of FK506.

[0095] Example 7: Application of Streptomyces pyogenes M4018 / CN overexpressing NADH kinase element sPos5 in increasing oxytetracycline production

[0096] The recombinant strains M4018 / CN, M4018 / C of Streptomyces pyogenes obtained in Example 2 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 250rpm for 24h. Then, the spores were transferred to Streptomyces pyogenes fermentation medium with an inoculum size of 5%, and cultured at 28°C and 250rpm for 8 days.

[0097] 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.

[0098] The composition of the seed culture medium: 30g / L starch, 3g / L soybean cake powder, 4g / L ammonium sulfate, 5g / L calcium carbonate, 4g / L corn steep liquor, 5g / L sodium chloride, 0.15g / L potassium dihydrogen phosphate, and the balance is water.

[0099] The composition of the fermentation medium is: 150g / L starch, 20g / L soybean cake powder, 14g / L ammonium sulfate, 14g / L calcium carbonate, 4g / L corn steep liquor, 4g / L sodium chloride, 0.1g / L potassium dihydrogen phosphate, 10μg / L cobalt chloride, 0.1%-0.2% amylase, and the balance is water.

[0100] Detection method of oxytetracycline: take 1.5mL of fermentation broth, acidify it with 9mol / L hydrochloric acid to pH 1.5 to 1.7, centrifuge and take the supernatant through a membrane for HPLC detection. Detection method: use SB-C18 (250mm×4.6mm, Agilent, America) chromatographic column, the volume ratio of the mobile phase is water: methanol: acetonitrile: 2mM phosphoric acid solution = 60:10:20:10, the flow rate is 1.0mL / min, the detection wavelength is 350nm, and the injection volume is 10μL.

[0101] The test results of oxytetracycline production are as follows Figure 7 As shown, compared with the starting strain M4018, the oxytetracycline production of the recombinant strain M4018 / CN of Streptomyces pyrrosinum increased by 56.43% to 2.04 g / L, and the empty control strain M4018 / C had no effect on the oxytetracycline production.

[0102] Example 8: Application of Streptomyces roseosporus NRRL 11379 / CN overexpressing NADH kinase element sPos5 in increasing daptomycin production

[0103] The recombinant strains NRRL 11379 / CN, NRRL 11379-C of Streptomyces roseosporus obtained in Example 2 and the original strain NRRL 11379 were inoculated on DAI solid culture medium, cultured at 28°C for 7 days, scraped and cultured in a primary seed culture medium of Streptomyces roseosporus at 28°C and 250rpm for 60h, then transferred to a secondary seed culture medium with an inoculum size of 5%, cultured at 28°C and 250rpm for 36h, and then transferred to a fermentation culture medium of Streptomyces roseosporus with an inoculum size of 5%, cultured at 28°C and 250rpm for 10 days, and 0.5mL of 2% (W / V) sterile sodium decanoate solution was added every 12h starting from 48h.

[0104] The spore-forming medium was DAI medium, and the 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.

[0105] The composition of the seed culture medium: 5g / L glucose, 15g / L dextrin, 5g / L bacterial peptone, 5g / L yeast extract powder, 5g / L peanut cake powder, 0.5g / L dipotassium hydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, 0.2g / L calcium carbonate, and the balance is water.

[0106] 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.

[0107] Daptomycin detection method: take 1 mL of fermentation broth, centrifuge twice at 13000 rpm, 4°C for 15 minutes, take the supernatant and pass it through a membrane for HPLC detection. Detection method: use 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.

[0108] The test results of daptomycin production are as follows Figure 8 As shown, compared with the starting strain NRRL 11379, the daptomycin production of the recombinant strain NRRL 11379 / CN of Streptomyces roseosporus increased by 35.06% to 329.57 mg / L, and the empty control strain NRRL 11379 / C had no effect on the daptomycin production.

[0109] Example 9: Application of Streptomyces venezuelae ISP5230 / CN overexpressing NADH kinase element sPos5 in improving the yield of jedomycin

[0110] The recombinant strains ISP5230 / CN and ISP5230 / C of Streptomyces venezuelae obtained in Example 2 and the original strain ISP5230 were inoculated on MYM solid culture medium, respectively, and cultured at 30°C for 5 days. Spores were scraped and inoculated on seed culture medium, and cultured at 30°C and 200 rpm for 20 hours. Then, the spores were transferred to fermentation medium and cultured at 30°C and 200 rpm for 48 hours.

[0111] 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.

[0112] The seed culture medium was liquid MYM medium.

[0113] 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), 4.5mL0.2g / 100mLFeSO 4 7H 2 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 of 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 (9mM) contains 10.5g K 2 HPO 4 and 4.5 g KH 2 PO 4 .

[0114] Jedomycin detection method: take 1mL 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. The detection method is: use an SB-C18 (250mm×4.6mm, Agilent, America) chromatographic column, the mobile phase is A: water, B: acetonitrile + 0.1% trifluoroacetic acid, gradient elution, flow rate 1.0mL / min, detection wavelength 316nm, injection volume 20μL.

[0115] The test results of jedomycin production are as follows Fig. 9 As shown, compared with the starting strain ISP5230, the jedomycin production of the recombinant strain ISP5230 / CN of Streptomyces venezuelae increased by 23.43% to 308.42 μg / mL, and the empty control strain ISP 5230 / C had no effect on the jedomycin production.

[0116] Example 10: Application of Streptomyces albus IPPDNR / CN overexpressing NADH kinase element sPos5 in improving daunorubicin production

[0117] The recombinant strains of Streptomyces albus IPPDNR / CN, IPPDNR / C obtained in Example 2 and the starting strain IPPDNR were inoculated on MS solid culture medium, respectively, and cultured at 30°C for 3 days. 1 cm×2 cm spores 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 inoculation amount was transferred to a secondary seed culture medium at 5%, and cultured at 30°C and 220 rpm for 24 hours. The inoculation amount was then transferred to a fermentation culture medium at 10%, and cultured at 30°C and 250 rpm for 10 days.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] Daunorubicin detection method: take 1mL of fermentation broth, add 9mL of methanol, let it stand for 12h and then centrifuge, take the supernatant and pass it through a membrane for HPLC detection. Detection method: use SB-C18 (250mm×4.6mm, Agilent, America) chromatographic column, XDB-C18, mobile phase A: 0.01M ammonium acetate aqueous solution (pH=4.2), mobile phase B: acetonitrile, flow rate 1.0mL / min, detection wavelength is 254nm, column temperature 37℃, gradient washing, injection volume 20μL.

[0122] The test results of daunorubicin production are as follows Fig.10 As shown, compared with the starting strain IPPDNR, the daunorubicin production of the recombinant strain IPPDNR / CN of Streptomyces albus increased by 30.52% to 31.82 μg / mL, and the empty control strain IPPDNR / C had no effect on the daunorubicin production.

[0123] Example 11: Application of Saccharopolyspora spinosa NRRL 18395 / CN overexpressing NADH kinase element sPos5 in increasing the production of spinosad

[0124] The recombinant strains of Saccharopolyspora spinosa NRRL 18395 / CN, NRRL 18395 / C and the starting strain NRRL 18395 obtained in Example 2 were inoculated on spore-forming solid culture medium and cultured at 30°C for 7 days. Spores were scraped and inoculated into Saccharopolyspora spinosa seed culture medium, cultured at 28°C, 250rpm for 72h, and then transferred to Saccharopolyspora spinosa fermentation medium at 28°C, 250rpm with an inoculum amount of 10% and cultured for 8 days.

[0125] The composition of the spore production medium is as follows: 20 g / L whole milk powder, 3 g / L yeast extract, 5 g / L glucose, 20 g / L agar, and the balance is water.

[0126] The composition of the seed culture medium is as follows: 10 g / L whole milk powder, 10 g / L glucose, 5 g / L yeast extract powder, and 5 g / L peptone.

[0127] The composition of the fermentation medium is as follows: whole milk powder 10 g / L, glucose 100 g / L, yeast extract powder 5 g / L, peptone 10 g / L, soybean oil 10 g / L, K 2 HPO 4 1 g / L, CaCO 3 5 g / L.

[0128] Spinosad detection method: 2 mL of fermentation broth was added with 4 mL of anhydrous methanol, ultrasonically treated for 1 h, centrifuged at 12000 rpm for 10 min, and the supernatant was directly used for HPLC analysis. HPLC analysis conditions: Agilent Zorbax Eclipse XDB-C8 (4.6 mm × 150 mm, 5 μm; item number: 993967-906) chromatographic column, detection wavelength 246 nm; the volume ratio of the mobile phase was methanol: acetonitrile: water (containing 0.05% ammonium acetate) = 45:45:10; flow rate 1.0 mL / min, injection volume 20 μL.

[0129] The test results of spinosad production are as follows Fig.11 As shown, compared with the starting strain NRRL 18395, the production of spinosad in the recombinant strain NRRL 18395 / CN of Saccharopolysaccharides increased by 30.67% to 37.64 mg / L, and the empty control strain NRRL 18395 / C had no effect on the production of spinosad.

[0130] 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 NADH kinase element sPos5 in increasing the yield of secondary metabolites of actinomycetes, characterized in that: The nucleotide sequence of the NADH kinase element sPos5 is shown in SEQ ID NO.1, and the secondary metabolite is a polyketide compound, a polyether compound or a non-ribosomal peptide compound.

2. Use of a recombinant vector containing the NADH kinase element sPos5 of claim 1 in increasing the yield of secondary metabolites of actinomycetes, characterized in that: The secondary metabolites are polyketide compounds, polyether compounds or non-ribosomal peptide compounds.

3. The use according to claim 2, characterized in that: The starting vector of the recombinant vector is pSET152, the promoter actⅡ-orf4p fragment is amplified using the genome of Streptomyces coelicolor as a template, and the promoter actⅡ-orf4p fragment and the NADH kinase element sPos5 fragment are integrated into the starting vector to obtain the recombinant vector.

4. The use according to claim 3, characterized in that: The nucleotide sequences of the upstream primer and the downstream primer used to amplify the promoter actⅡ-orf4p fragment are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

5. The use according to claim 3 or 4, characterized in that: The recombinant vector is used for increasing the yield of actinomycetophthrin produced by fermentation of Streptomyces coelicolor.

6. The use according to claim 2, characterized in that: The starting vector of the recombinant vector is pSET152::ermEp*, and the recombinant vector is obtained by integrating the NADH kinase element sPos5 fragment into the starting vector.

7. The use according to claim 6, characterized in that: The recombinant vector is used to increase the yield of milbemycin or nanchangmycin in Streptomyces bingchenggensis, increase the yield of avermectin in Streptomyces avermitilis, increase the yield of FK506 in Streptomyces tsukubaensis, increase the yield of oxytetracycline in Streptomyces rimosus, increase the yield of daptomycin in Streptomyces roseosporus, increase the yield of daptomycin in Streptomyces venezuelae, increase the yield of daunorubicin in Streptomyces albus, or increase the yield of spinosad in Saccharopolysporaspinosa.

8. Use of a recombinant bacterium containing the recombinant vector of claim 3 in increasing the yield of actinomycetophthrin produced by fermentation of Streptomyces coelicolor, characterized in that: The recombinant bacteria is obtained by taking Streptomyces coelicolor M145 as the starting strain and introducing the recombinant vector described in claim 3 into the starting strain.

9. Use of a recombinant bacterium containing the recombinant vector according to claim 6 in increasing the yield of secondary metabolites of actinomycetes, characterized in that: The recombinant bacteria are obtained by introducing the recombinant vector described in claim 6 into the starting strain, using Streptomyces BC-101-4, Streptomyces avermitilis S0, Streptomyces tsukuba NRRL18488, Streptomyces crassifolius M4018, Streptomyces roseosporus NRRL 11379, Streptomyces venezuelae ISP5230, Streptomyces albus IPPDNR or Saccharopolyspora spinosa NRRL 18395 as the starting strain.

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 avermitilis S0, the recombinant bacteria is used to increase the production of avermectin; when the starting strain is Streptomyces tsukuba NRRL 18488, the recombinant bacteria is used to increase the production of FK506; when the starting strain is Streptomyces crassa M4018, the recombinant bacteria is used to increase the production of oxytetracycline; when the starting strain is Streptomyces roseosporus 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 daunorubicin; when the starting strain is Streptomyces albus IPPDNR, the recombinant bacteria is used to increase the production of daunorubicin; when the starting strain is Saccharopolyspora spinosa NRRL 18395, the recombinant bacteria is used to increase the production of spinosad.

Images