A transporter protein T6417 and its application in improving the yield of Streptomyces natural products

Abstract

A transporter protein T6417 and its application in improving the yield of Streptomyces natural products belong to the field of genetic engineering technology. In order to improve the yield of Streptomyces natural products, based on the analysis of the transcriptome data of Streptomyces caniferus NEAU6, this invention screened transporter proteins related to yield and the timing of antibiotic production, and obtained the candidate transporter protein T6417 through functional annotation analysis; then the expression of T6417 was adapted using different promoters, and it was found that overexpressing the transporter protein T6417 in Streptomyces caniferus, Streptomyces bingchenggensis or Streptomyces avermitilis can improve the yield of natural products. This invention provides valuable biosynthetic elements for the efficient biomanufacture of Streptomyces.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a transporter protein T6417 and its application in improving the yield of Streptomyces natural products. Background Art

[0002] Microbial natural pesticides refer to pesticides with secondary metabolites of microorganisms as active ingredients. 90% of the active substances used in fields such as medicine and agriculture are derived from secondary metabolites synthesized by actinomycetes, such as macrolide antibiotics, aminoglycoside antibiotics, tetracyclines, nucleoside antibiotics, etc. More than 50% of Streptomyces can produce antibiotics (SAITOS, KATO W, IKEDA H, et al. Discovery of “heat shock metabolites” produced by thermotolerant actinomycetes in high-temperature culture [J]. The Journal of Antibiotics, 2020, 73(4): 203-10.). As a dominant group of microorganisms for producing bioactive substances such as antibiotics and biological pesticides, Streptomyces has broad development prospects (DONG Lei, HAN Jiarui, LI Shuai, et al. The latest research progress of Streptomyces [J]. Acta Microbiologica Sinica, 2023, 63(05): 1815-32.). However, under natural conditions, the biosynthesis efficiency of Streptomyces is relatively low, and its ability to produce secondary metabolites cannot meet the large-scale industrial and agricultural applications. Therefore, engineering transformation of Streptomyces is of great significance for promoting the efficient biosynthesis of secondary metabolites and facilitating green bio-manufacturing.

[0003] As an important producer strain of natural product pesticides, Streptomyces genomes contain rich resources of transport proteins. Transport proteins account for 12.1% of the proteome in Streptomyces coelicolor; 13.7% in Streptomyces griseus; and 12.9% in Streptomyces avermitilis (ZHOU Z, SUN N, WU S, et al. Genomic data mining reveals a rich repertoire of transport proteins in Streptomyces [J]. BMC Genomics, 2016, 17(S7)). The transport system of Streptomyces is also the main reason why the bacteria can be used as industrial production strains. Transport proteins can not only directly participate in the antibiotic transport process, but also work together with the regulatory system to ensure the stable growth and development of the bacteria themselves (ZHANG Guofeng, LIANG Dongmei, QIAO Jianjun, Caiyin Qinggele. Research progress on the transport system of Streptomyces [J]. Microbiology China, 2020, 47(11): 3689-98). Therefore, transporter engineering based on the excavation and engineering transformation of the transport system is an effective strategy for constructing high-yield industrial production strains. Summary of the Invention

[0004] To improve the yield of natural products of Streptomyces, based on the analysis of the transcriptome data of Streptomyces caniferus NEAU6, this invention screened transport proteins related to yield and the timing of production, and obtained the candidate transport protein T6417 through functional annotation analysis; then different promoters were used to adapt the expression of T6417, and it was found that overexpressing the transport protein T6417 in Streptomyces caniferus, Streptomyces bingchenggensis or Streptomyces avermitilis could increase the yield of natural products.

[0005] To solve the above technical problems and achieve the corresponding technical effects, this invention provides the following technical solutions:

[0006] The first object of this invention is to provide a transport protein T6417, and the amino acid sequence of the transport protein T6417 is shown as SEQ ID NO.1.

[0007] The second object of this invention is to provide a gene encoding the above transport protein T6417, and the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0008] The third object of this invention is to provide a recombinant vector, and the recombinant vector contains the above gene.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned transporter T6417, the above-mentioned gene or the above-mentioned recombinant vector in constructing recombinant Streptomyces caniferus producing glabervictin.

[0010] The fifth object of the present invention is to provide the use of the above-mentioned transporter T6417, the above-mentioned gene or the above-mentioned recombinant vector in constructing recombinant Streptomyces bingchenggensis producing milbemycin.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned transporter T6417, the above-mentioned gene or the above-mentioned recombinant vector in constructing recombinant Streptomyces avermitilis producing avermectin.

[0012] The seventh object of the present invention is to provide a recombinant bacterium, which contains the above-mentioned gene or the above-mentioned recombinant vector.

[0013] The eighth object of the present invention is to provide a method for constructing the above-mentioned recombinant bacterium, and the construction method includes the following steps: constructing a recombinant plasmid containing the gene encoding transporter T6417, transforming the recombinant plasmid into Escherichia coli, and introducing the recombinant plasmid into the starting Streptomyces through conjugation transfer.

[0014] In one embodiment of the present invention, the promoter used in constructing the recombinant plasmid is P ermE* 、P hrdB 、P 1140 、P 7518 or P 7518-ant ,and the nucleotide sequences of P ermE* 、P hrdB 、P 1140 、P 7518 and P 7518-ant are shown as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27 respectively.

[0015] In one embodiment of the present invention, the starting Streptomyces is Streptomyces caniferus, Streptomyces bingchenggensis or Streptomyces avermitilis.

[0016] Advantages of the present invention:

[0017] Based on the analysis of the transcriptome data of Streptomyces caniferus NEAU6, the present invention screens the transporters related to yield and the timing of antibiotic production, and obtains the candidate transporter T6417 through functional annotation analysis; then different promoters (PermE* , P hrdB , P 1140 , P 7518 and P 7518-ant ) adapted the expression of T6417 and found that overexpressing the transporter T6417 in Streptomyces cinereus, Streptomyces bingchenggensis or Streptomyces avermitilis could increase the yield of natural products. Among them, using Streptomyces cinereus NEAU6 as the starting strain, the recombinant strains N6-P ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417 had the avermectin yields of 665 mg / L, 658 mg / L, 705 mg / L, 673 mg / L, and 903 mg / L respectively. Compared with the starting strain NEAU6, the avermectin yields of the 5 recombinant strains increased by 27.2%, 22.4%, 31.3%, 25.3%, and 77.4% respectively; using Streptomyces bingchenggensis BC-101-4 as the starting strain, the recombinant strain BC-P 7518-ant -6417 had the milbemycin A4 yield of 1259 mg / L, and the milbemycin A4 yield increased by 13.2% compared with the starting strain Streptomyces bingchenggensis BC-101-4; using Streptomyces avermitilis S0 as the starting strain, the recombinant strain S0-P 7518-ant -6417 had the avermectin yield of 3 g / L, and the avermectin B1a yield increased by 50% compared with the starting strain Streptomyces avermitilis S0. The present invention provides valuable biosynthetic elements for the efficient biomanufacturing of streptomyces. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the result diagram of the structure prediction of the transporter T6417;

[0019] Figure 2 is the result diagram of the transmembrane prediction of the transporter T6417;

[0020] Figure 3 is the schematic diagram of the construction of the recombinant vector; among them, Figure 3 A in is the schematic diagram of the construction of the recombinant vectors P ermE* -6417, P hrdB -6417, P 1140 -6417 and P 7518 -6417, Figure 3 B in is the schematic diagram of the construction of the recombinant vector P 7518-ant -6417;

[0021] Figure 4 For Streptomyces cinereus NEAU6 and recombinant strain N6-P ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant Detection result graph of the yield of -6417 avermectin

[0022] Figure 5 For Streptomyces bc101-4 and recombinant strain BC-P 7518-ant Detection result graph of the yield of -6417 milbemycin

[0023] Figure 6 For Streptomyces avermitilis S0 and recombinant strain S0-P 7518-ant Detection result graph of the yield of -6417 avilamycin Specific implementation manners

[0024] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with specific implementation manners 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 not for limiting the scope of the present invention. The embodiments mentioned below are only some embodiments of the present invention rather than all embodiments. In the art, if other technical personnel do not make creative efforts, the embodiments obtained by them are protected by the present invention.

[0025] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, culture media and instruments used are all conventional materials, reagents, culture media and instruments in the art unless otherwise specified, and those skilled in the art can obtain them through commercial channels. The molecular biology experimental operations such as PCR amplification, restriction enzyme digestion and ligation, and transformation involved in the present invention are all conventional experimental operations in the art or can be carried out according to the product instructions of the corresponding reagents unless otherwise specified.

[0026] The strain information involved in the following embodiments is shown in Table 1; the plasmid information involved is shown in Table 2; the primer information involved is shown in Table 3.

[0027] Table 1 Strain information involved in the embodiments

[0028]

[0029] Table 2 Plasmid information involved in the embodiments

[0030]

[0031] Table 3 Primer information involved in the embodiments

[0032]

[0033]

[0034] Note: P ermE* -F / R is used for amplifying the P ermE* fragment, and P hrdB -F / R is used for amplifying the P hrdB fragment, and P 1140 -F / R is used for amplifying the P 1140 fragment, and P 7518 -F / R is used for amplifying the P 7518 fragment, ant-F / R is used for amplifying the amplifier ant fragment, ermE*-6417-F / R, hrdB-6417-F / R, P 1140 -6417-F / R, P 7518 -6417-F / R and P 7518-ant -6417-F / R are both used for amplifying the scn6417 fragment.

[0035] The strain sources used in the present invention are as follows:

[0036] Streptomyces caniferus NEAU6 is recorded in the Chinese patent with the application number 201910189570.2 and the invention title of "A Streptomyces Strain and Its Application", and the public can obtain it from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0037] 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 induced mutants. World J Microbiol Biotechnol 25: 1051-1056.

[0038] Streptomyces avermitilis S0 is disclosed in the patent with the application number CN202310480048.6 and the invention title of "A Sugar Transporter TP6568 and Its Application in Transforming High-Yield Streptomyces".

[0039] The culture medium involved in the present invention is as follows:

[0040] The solid medium for the growth and fermentation of Streptomyces cinereogriseus is the YMS solid medium, and its composition is as follows: 4 g / L yeast extract powder, 10 g / L malt extract powder, 4 g / L soluble starch, 20 g / L agar, with the balance being water, and the pH is 7.4.

[0041] The composition of the seed medium for Streptomyces cinereogriseus is: 20 g / L sucrose, 20 g / L maltodextrin, 60 g / L soybean powder, 3 g / L calcium carbonate, with the balance being water, and the pH is 7.0.

[0042] The composition of the fermentation medium for Streptomyces cinereogriseus is: 10 g / L sucrose, 60 g / L maltodextrin, 40 g / L soybean powder, 4 g / L calcium carbonate, with the balance being water, and the pH is 7.2.

[0043] The solid medium for the growth and fermentation of Streptomyces bingchenggensis is the SKYM medium, and the specific composition is as follows: 4 g / L sucrose, 1 g / L skim milk powder, 2 g / L yeast extract powder, 5 g / L malt extract powder, 20 g / L agar powder, with the balance being water.

[0044] The composition of the seed medium for Streptomyces bingchenggensis: 5 g / L yeast extract powder, 10 g / L sucrose, 1 g / L skim milk powder, 3.5 g / L bacteriological peptone, 0.5 g / L dipotassium hydrogen phosphate, with the balance being water.

[0045] The composition of the fermentation medium for Streptomyces bingchenggensis: 80 g / L sucrose, 20 g / L soybean cake powder, 1 g / L skim milk powder, 0.1 g / L ferrous sulfate, 1 g / L dipotassium hydrogen phosphate, 3 g / L calcium carbonate, with the balance being water.

[0046] The solid medium for the growth and fermentation of Streptomyces avermitilis is the GYM solid medium, and the specific composition is as follows: 4 g / L soluble starch, 4 g / L yeast extract powder, 10 g / L malt extract powder, 5×10 -5 g / L cobalt chloride hexahydrate, 20 g / L agar powder, with the balance being water.

[0047] The composition of the seed medium for Streptomyces avermitilis: 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, with the balance being water.

[0048] The composition of the fermentation medium for Streptomyces avermitilis: 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, with the balance being water.

[0049] The tool enzymes, kits and other reagents selected in the present invention are as follows:

[0050] The DNA polymerase 2×SuperNova PCR Mix (Dye) was purchased from GenStar, the Gibson assembly enzyme ExnaseMultiS was purchased from Vazyme, the restriction endonucleases were purchased from Takara and NEB, the agarose gel recovery kit was purchased from OMEGA, the plasmid extraction kit was purchased from Axygen, the kit for RNA extraction was purchased from Beijing ComWin Biotech Co., Ltd., and the reagents used for RNA purification, reverse transcription and fluorescence quantification were purchased from Thermo Fisher. The chromatographically pure methanol and acetonitrile reagents for HPLC were both from MREDA.

[0051] Operations such as polymerase chain reaction (PCR), agarose gel recovery, and Gibson assembly involved in the present invention were all carried out according to the instructions of the reagents and kits.

[0052] The amino acid sequence of the efflux transporter T6417 in the present invention is shown as SEQ ID NO.1, and the nucleotide sequence of its encoding gene scn6417 is shown as SEQ ID NO.2. The promoters P ermE* , P hrdB , P 1140 , P 7518 , and the nucleic acid sequence of the amplifier fragment ant are shown as SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27 respectively.

[0053] SEQ ID NO.1:

[0054] VAPTTTPEKSEIPPRSRIFADLTPLRLSPDYRRLWCGNTVSWMGQQMTALAVSLQVYTLTHSTFAVGLVGLCSLVPLVVFGLYGGAIADTVDRRKLGLYSAAGATVMSLTLATAALAGYHRVWLLYTVVAFQAVCFAMNSPARSSMIPRLLPPEQLPAANALNSLTSNLGLMGGPMLGGVIVGLWGFQAAYLIDVVAFSGSLYAMWRLPSMRPDQGEGPRRRASVLDGLRFLATRPNLRMTFFSDLAAMVLAQPRALFPAVAVLWFGGDAKTVGLLVAAPAVGAVLGGLFSGWLGGIRRHGLAILIAVAAWGAAIACFGLSRHLWLGLFFLAVAGCADTVSMVFRSTMLQAATPDAMRGRLQGVFIVVVAGGPRLGDFLAGSVADLTSPATAVLGGGLACVLVVTGLGLGRRAFARYDARDPQP

[0055] SEQ ID NO.2:

[0056]

[0057] SEQ ID NO.23:

[0058] ctctagtatgcatgcgagtgtccgttcgagtggcggcttgcgcccgatgctagtcgcggttgatcggcgatcgcaggtgcacgcggtcgatcttgacggctggcgagaggtgcggggaggatctgaccgacgcggtccacacgtggcaccgcgatgctgttgtgggcacaatcgtgccggttggtaggatccacat

[0059] SEQ ID NO.24:

[0060] ccgccttccgccggaacggcggggtccgggcacgccaaacccctcctgtggctgtggccggccaccgccgtcaccttcggaccccgtggagccgctcccggttccacggggtccgaaggtgtgatgagcaggctgcgccttcctcgcgcggccgcaaggtacgagttgatgaccttgtttatccgcatctgaccaattttgatcgcttacggggtgtgactcgggccacgcggattgggcgtaacgctcttgggaacaacacgatgacctaagaggtgacagccgcggagggaatacggacgccgttcacggcgctgtgcatctccccggcccgcccgcaccgtcggcccattcccaagccggtggtcggcccctgtccgccgtggacggggccggaagccgtttttcaacgttccgagaggttgttc

[0061] SEQ ID NO.25:

[0062] ggacacggcacaggcgagaacgccgtcgatggcctggcggggagaggggaacgggcccttcccgtcctcgccctggagggggtagacgatcaccccgatcccgtggtcggcgccgacgcgggccgctccgctgtgcacatcggcgaaatgggggttcgtcagggacggcacgaccagcatgaccgtgccggtccggcctaatcgcagactgcgcgccgaaagattgagccgataacccatgtccgccgccgtatccagaaccttcttcgccatttttgccgagacgcggccacgccactttcccgccatcaccaacgagacggtggcctgtgacacgccaagcgtccgtgccaagtccttacttgtcatttgaggcgcgttaaccggtgcgttccctgccctgtggagtccttcagacaacggtccctcagctcttatgcggactacggaagttcagctttcaccagatcttgcgccggacatcatacgtatgatctggtagcggacatcatacgtatgatgcggttgctcaaccagagataacagtcagcgacattcggcgcatggagacctca

[0063] SEQ ID NO.26:

[0064] gatccgctaccagctgctcaaccagccggggctcgacgaccggcaggagacctggtcggcgcggcacttcaccgacacgccctggatgaatcgcgacaaagtcaccgcctcgtgggggcactacctgggctccacgcccgccacgccgtacgccgccccggcgcgggccaccgacctgtccggcctgccacccgcctacatcgccaccgcggaactctgtccgaaccgcgacgaagacatcacctacgcgctacgactgttgcaggccggcgtctccgtcgaactgcaccagtggtccggcacgttccacgggtcgcaggcgatcctgtccgccgaggtgtcccagcggcagatcgccgaactcgccgcagtcctgcgccgtgccctggccgattgaggcggctgcggctgcggctcggctgcggtcgcggccgagccggctggtgacccccatgccgcaacacacccagatgaaagtccaccgcaggaaggacattcccgc

[0065] SEQ ID NO.27:

[0066] atgaacaccgcgcacgaactgcccgcccccgtggaccggttgctgccgcgggcgagggcgggcgacgagcaggcgatgaacgacctgctgctgcacatcaccccgtacgtcacccgtgtctgccgctcggtcgcccacgacaacggctccgacgcccggcaggaggccctcctcgcgatctaccggggcctgccggggctgcgggagcccgccgccttctacgggtgggtccgctcggtcaccgtgcgggaggcggtgcgcacggccaggcgcctgggccgcgagacgacgagccccgaggtggactcgcgccacgaaccccactcactcgacgccgtccacatcaacgacgtcctggaccgcatgtccgaggcccaccgccaggtgctgacccttcgcgtgtacggcctgaacgaggtggagatggccgagaccctcgccctccccgtcggcacggtccgctcccggctccaccgggcccgccgccgcttccaggaggcatggcagcccaccgacgactaaaacaacaagaaggaaccagggccgcttcaagagaagcggccctggttctttcttttttactagacaacaagaaggaaccagggccgcttcaagagaagcggccctggttctttcttttttactagagtcgacctgcagcccaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatccctcccgatccgccgcgtgacgagcgtcaccacggggccgggggattgccgccgcctcctcgcgcctcttcctctgcgacaacacacgtgtgtggccgggaccggcggacggccgcggctgagtgaagaggagaaggc

[0067] The detection method involved in the present invention is as follows:

[0068] Detection method of avermectin: Take 0.25 mL of the fermentation broth or the supernatant of the fermentation broth of Streptomyces avermitilis and mix it thoroughly with 1.20 mL of methanol. Ultrasonically treat the sample for 40 min to extract the total avermectin and extracellular avermectin in the fermentation broth. Subsequently, centrifuge the preliminarily treated sample at a high speed of 12000 rpm for 15 min, aspirate the upper avermectin extract, filter it through a 0.22 μM organic filter membrane, and then perform liquid phase detection. 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 a methanol solution with a volume fraction of 90%, the flow rate is 1.0 mL / min, and the column temperature is 35°C.

[0069] Detection method of milbemycin: Take 0.5 mL of the fermentation broth of Streptomyces hygroscopicus var. jinggangensis and mix it thoroughly with 1.5 mL of ethanol. Invert and shake the treated sample for 30 min to extract milbemycin in the fermentation broth. Subsequently, centrifuge the preliminarily treated sample at a high speed of 12000 rpm for 15 min, aspirate the upper milbemycin extract, filter it through a 0.22 μM organic filter membrane, and then perform liquid phase detection. The detection instrument is Agilent 1260 high performance liquid chromatography (HPLC), the chromatographic column is a C18 column (Zorbax, 4.6 mm × 250 mm, 5 μm), the detection wavelength is 242 nm, the injection volume is 20 μL, the flow rate of the mobile phase is 1.0 mL / min. The specific chromatographic conditions are as follows: within 0 - 15 min, mobile phase A (methanol) elutes from 0% to 100%, and mobile phase B (volume ratio: acetonitrile:methanol:water = 7:2:1) gradient elutes from 100% to 0%; from 15 - 17 min, mobile phase A maintains 100%; from 17 - 25 min, mobile phase A (methanol) elutes from 100% to 0%, and mobile phase B gradient elutes from 0% to 100%; from 25 - 27 min, mobile phase B maintains 100%, and the column temperature is 28°C.

[0070] Detection method of gorizomycin: Collect 0.4 mL of the fermentation broth of Streptomyces caesius, add 1.60 mL of ethanol, ultrasonically treat it for 60 min to extract gorizomycin in the fermentation broth, and centrifuge it at 12000 rpm for 10 min. Take the supernatant, filter it through a 0.22 μM organic filter membrane, and then perform liquid phase detection. The detection instrument is Agilent 1260 high performance liquid chromatography (HPLC), the chromatographic column is a C18-aq column (ZorbaxSB-Aq, 4.6 mm × 250 mm, 5 μm), the mobile phase is acetonitrile with a volume fraction of 11% and ultrapure water with a volume fraction of 89%, the flow rate is 0.8 mL / min, the detection wavelength is 260 nm, and the column temperature is 28°C.

[0071] Example 1: Obtaining of transporter T6417

[0072] Based on the time-series transcriptome data of Streptomyces tenebrarius NEAU6 and NEAU6-D39, genes related to secondary metabolite production and time series were screened, and the candidate transporter T6417 was obtained through functional annotation analysis.

[0073] The amino acid sequence of transporter T6417 is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene scn6417 is shown in SEQ ID NO.2. AlphaFold 2 and DeepTMHMM 1.0 (https: / / services.healthtech.dtu.dk / services / DeepTMHMM-1.0 / ) were used to predict the three-dimensional structure and transmembrane domain of T6417, and the results are shown in Figure 1 and Figure 2 respectively. As can be seen from Figure 1 , the N-terminus and C-terminus of T6417 are on the same side, and the sequence structure shows multiple transmembrane helix repeats, presenting a unique "MFS Fold" in the secondary domain. Therefore, it is speculated that T6417 is a transporter of the MFS superfamily (Deng Dong, Yan Ning. Structural basis and transport mechanism of MFS superfamily transporters [J]. Chinese Science Bulletin, 2015, 60: 720–728.). In addition, Figure 2 the transmembrane domain prediction results also show that T6417 has the structural characteristics of multiple transmembrane repeats.

[0074] Example 2: Construction of recombinant vectors overexpressing T6417 containing different promoters

[0075] In the life cycle of Streptomyces, the biosynthesis of natural products generally occurs in the late growth stage. Therefore, in the present invention, the expression of T6417 was adapted by using different promoters. The candidate promoters include the constitutive promoters P ermE* and P hrdB , and the time-series promoters P 1140 , P 7518 and P 7518-ant . Among them, the time-series promoter P 1140 is the promoter with the highest expression intensity in the GV biosynthesis gene cluster of Streptomyces tenebrarius NEAU6. P 7518 was screened from the whole genome of Streptomyces tenebrarius NEAU6. The specific screening was as follows: FPKMD3 < 300 and FPKMD5 > 500, and then the promoter P 7518 was obtained through heat map clustering. The time-series promoter P 75118-ant is a recombinant promoter carrying a gene amplifier for promoter P 7518 , which can amplify the expression intensity of promoter P 7518 while expressing in time series.

[0076] The plasmid pSET152 was double-digested with restriction endonucleases XbaI and EcoRI to obtain the linear vector backbone LpSET152; the primers P ermE* -F / R, P hrdB -F / R, P 1140 -F / R, P 7518 -F / R were used to amplify the P ermE* , P hrdB、 P 1140 , P 7518 promoter fragments respectively, and the primer ant-F / R was used to amplify the amplifier fragment; the primers ermE-6417-F / R, hrdB-6417-F / R, P 1140 -6417-F / R, P 7518 -6417-F / R, P 7518-ant -6417-F / R were used to amplify the scn6417 fragments of different promoter vectors using the NEAU6 genome as a template. Subsequently, the promoter fragments, amplifier fragment and scn6417 fragment were assembled by Gibson assembly, and the assembly system was introduced into Escherichia coli JM109 by heat shock transformation. Then, the Escherichia coli was inoculated into LB liquid medium containing apramycin (final concentration 100 μg / mL) and cultured overnight at 37 °C. The plasmid was extracted using a plasmid extraction kit to obtain the recombinant plasmids P ermE* -6417, P hrdB -6417, P 1140 -6417, P 7518 -6417, P 7518-ant -6417, and then agarose gel electrophoresis and sequencing verification were performed. The schematic diagram of the construction of the recombinant vector is as shown in Figure 3 .

[0077] Example 3: Construction of recombinant strains of overexpressing T6417 with different promoters

[0078] The successfully verified recombinant plasmids P ermE* -6417, P hrdB -6417, P 1140 -6417, P 7518 -6417, P 7518-ant -6417 in Example 2 were respectively introduced into Escherichia coli ET12567(pUZ8002) by heat shock transformation. The obtained Escherichia coli was inoculated into LB liquid medium containing apramycin, kanamycin and chloramphenicol (final concentration 100 μg / mL each) for culture, and then the plasmid was introduced into Streptomyces glaucescens NEAU6 by conjugation transfer to obtain recombinant strains expressing T6417 with different promoters (N6-P ermE* -6417, N6-P hrdB-6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417).

[0079] According to the annotation of the transcriptome data of Streptomyces cinereus NEAU6, it was found that T6417 is a putative macrolide efflux protein. Therefore, Streptomyces bingchenggensis BC-101-4, the producer of milbemycin, and Streptomyces avermitilis S0, the producer of avermectin, were selected as examples to verify the effect of T6417 on improving the yield of polyketide natural products. Based on the previous data, the recombinant plasmid P 7518-ant -6417 was selected for subsequent verification.

[0080] The recombinant plasmid P 7518-ant -6417, which was verified successfully in Example 2, was introduced into Escherichia coli ET12567(pUZ8002) by heat shock transformation. The obtained Escherichia coli was inoculated into LB liquid medium containing apramycin, kanamycin, and chloramphenicol (final concentrations were all 100 μg / mL) for culture, and then the plasmid was introduced into Streptomyces bingchenggensis BC-101-4 and Streptomyces avermitilis S0 by conjugation transfer respectively to obtain the recombinant strains BC-P 7518-ant -6417 and S0-P 7518-ant -6417.

[0081] The specific operation method of the above conjugation transfer refers to the following literature:

[0082] HE H, YE L, LI C, et al. SbbR / SbbA, an Important ArpA / AfsA-Like System, Regulates Milbemycin Production in Streptomyces bingchenggensis[J]. Frontiersin Microbiology, 2018, 9.

[0083] SHI H, WANG J, LI S, et al. Coordinated regulation oftwo LacI familyregulators, GvmR and GvmR2, on guvermectin production in Streptomyces caniferus[J]. Synthetic and Systems Biotechnology, 2025, 10(1): 237-46.

[0084] Dong Z, Li L, Du G, et al. A previously unidentified sugar transporter for engineering of high-yield Streptomyces[J]. Applied Microbiology and Biotechnology, 2024, 108(1).

[0085] Example 4: Application of recombinant Streptomyces tenebrarius NEAU6 strains in improving the yield of avemectin

[0086] Inoculate 5 recombinant strains N6-P ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417 on sporulation medium YMS and culture for 7 days. Subsequently, use an inoculation spatula to scrape an appropriate amount of spores and inoculate them into the seed medium of Streptomyces tenebrarius, and culture at 28°C and 250 rpm for 24 h. Then, draw the seed liquid with an inoculation amount of 5% and inoculate it into the fermentation medium of Streptomyces tenebrarius, and culture at 28°C and 250 rpm for 7 days. Collect the fermentation broth and detect the yield of avemectin.

[0087] The results are as Figure 4 shown. The avemectin yields of recombinant strains N6-P ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417 reached 665 mg / L, 658 mg / L, 705 mg / L, 673 mg / L, and 903 mg / L respectively. Compared with the original strain NEAU6, the avemectin yields of the 5 recombinant strains increased by 27.2%, 22.4%, 31.3%, 25.3%, and 77.4% respectively, indicating that overexpression of T6417 by different promoters is helpful to improve the yield of avemectin produced by Streptomyces tenebrarius NEAU6 through fermentation.

[0088] Example 5: Application of recombinant Streptomyces bc101-4 strains in improving the yield of milbemycin

[0089] Inoculate the recombinant strain BC-P 7518-ant-6417 was inoculated on the sporulation medium SKYM and cultured at a constant temperature of 28°C for 9 days. About 1 square centimeter of spores was scraped and inoculated into the Streptomyces bingchenggensis seed medium, and cultured at 28°C and 250 rpm for 46 h. Then, the seed liquid was inoculated into the Streptomyces bingchenggensis fermentation medium at an inoculation amount of 6%, and cultured at 28°C and 250 rpm for 9 days. The fermentation broth was collected and the yield of milbemycin was detected.

[0090] The results are as Figure 5 shown. The milbemycin A4 yield of the recombinant strain BC-P 7518-ant -6417 reached 1259 mg / L, and the milbemycin A4 yield increased by 13.2% compared with the original strain Streptomyces bingchenggensis BC-101-4, indicating that the recombinant vector P 7518-ant -6417 is an effective biosynthetic element that can increase the yield of Streptomyces natural products and is of great significance for the synthesis of microbial natural products.

[0091] Example 6: Application of the recombinant strain of Streptomyces avermitilis S0 in increasing the yield of avermectin

[0092] The recombinant strain S0-P 7518-ant -6417 was inoculated on the sporulation medium GYM and cultured at a constant temperature of 28°C for 6 days. About 1 square centimeter of mycelium pieces was taken with an inoculation shovel and inoculated into the Streptomyces avermitilis seed medium, and cultured at 28°C and 250 rpm for 40 h. Then, the seed liquid was inoculated into the Streptomyces avermitilis fermentation medium at an inoculation amount of 6%, and cultured at 28°C and 250 rpm for 10 days. The fermentation broth was collected and the yield of avermectin was detected.

[0093] The results are as Figure 6 shown. The avermectin yield of the recombinant strain S0-P 7518-ant -6417 reached 3 g / L, and the avermectin B1a yield increased by 50% compared with the original strain Streptomyces avermitilis S0, indicating that the recombinant vector P 7518-ant -6417 is an effective biosynthetic element that can increase the yield of Streptomyces natural products and is of great significance for the synthesis of microbial natural products.

[0094] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of transporter T6417, transporter T6417-encoding gene or recombinant vector containing the transporter T6417-encoding gene in constructing recombinant Streptomyces caniferus producing avenaciolide, characterized in that, The amino acid sequence of the transporter T6417 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the transporter T6417 is shown in SEQ ID NO.

2.

2. Use of transporter T6417, the gene encoding transporter T6417, or a recombinant vector containing the gene encoding transporter T6417 in constructing a recombinant Streptomyces bingchenggensis producing milbemycin, characterized in that, The amino acid sequence of the transporter T6417 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the transporter T6417 is shown in SEQ ID NO.

2.

3. Use of transporter T6417, a gene encoding transporter T6417, or a recombinant vector containing the gene encoding transporter T6417 in constructing recombinant Streptomyces avermitilis producing avermectin, characterized in that, The amino acid sequence of the transporter T6417 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the transporter T6417 is shown in SEQ ID NO.

2.

4. Use of a recombinant bacterium overexpressing the gene encoding transporter T6417 in increasing the yield of avermectin, milbemycin or avilamycin, characterized in that, The nucleotide sequence of the gene encoding the transporter T6417 is shown in SEQ IDNO.

2. The starting strain of the recombinant bacterium is Streptomyces cinereogriseus, Streptomyces bingchenggensis or Streptomyces avermitilis. When the starting strain is Streptomyces cinereogriseus, the recombinant bacterium is used to increase the yield of glabervicin. When the starting strain is Streptomyces bingchenggensis, the recombinant bacterium is used to increase the yield of milbemycin. When the starting strain is Streptomyces avermitilis, the recombinant bacterium is used to increase the yield of avermectin.

5. The method for constructing the recombinant bacterium in the application according to claim 4, characterized in that, It includes the following steps: constructing a recombinant plasmid containing the gene encoding the transporter T6417, transforming the recombinant plasmid into Escherichia coli, and introducing the recombinant plasmid into the starting Streptomyces by conjugation transfer. The nucleotide sequence of the gene encoding the transporter T6417 is shown in SEQ ID NO.2, and the starting Streptomyces is Streptomyces cinereogriseus, Streptomyces bingchenggensis or Streptomyces avermitilis.

6. The construction method according to claim 5, characterized in that, The promoter used in the construction of the recombinant plasmid is P ermE* , P hrdB , P 1140 , P 7518 or P 7518-ant , P ermE* , P hrdB , P 1140 , P 7518 and P 7518-ant The nucleotide sequences of which are shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26 and SEQ ID NO.27 respectively.

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