Transport protein T6417 and application thereof in increasing yield of natural products of streptomyces

Abstract

The invention discloses a transporter T6417 and application thereof in increasing the yield of natural products of streptomyces, and belongs to the technical field of genetic engineering. In order to improve the yield of a streptomyces natural product, the method comprises the following steps: screening transporters related to the yield and a production time sequence on the basis of analysis on transcriptome data of streptomyces cerevisiae NEAU6, and carrying out function annotation analysis to obtain a candidate transporter T6417; then, different promoters are used for carrying out adaptation on the expression of the T6417, and it is found that the yield of natural products can be increased by overexpressing the transporter T6417 in streptomyces obscurvius, streptomyces bingchengensis or streptomyces avermitilis. The invention provides a valuable biosynthetic element for efficient biomanufacturing of streptomyces.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a transport protein T6417 and an application thereof in improving the yield of natural products of Streptomyces. Background Art

[0002] Microbial natural pesticides refer to pesticides with microbial secondary metabolites as active ingredients. 90% of the active substances used in the fields of medicine, agriculture, etc. are derived from secondary metabolites synthesized by actinomycetes, such as macrolide antibiotics, aminoglycoside antibiotics, tetracycline, nucleoside antibiotics, etc., and 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.). Streptomyces, as a dominant bacterial group for producing antibiotics, biological pesticides and other biologically active substances, has broad development prospects (Dong Lei, Han Jiarui, Li Shuai, et al. Latest research progress on Streptomyces [J]. Acta Microbiologica Sinica, 2023, 63 (05): 1815-32.). However, under natural conditions, the biosynthesis efficiency of Streptomyces is low, and its ability to produce secondary metabolites cannot meet the needs of a wide range of industrial and agricultural applications. Therefore, engineering modifications of Streptomyces are of great significance in promoting efficient biosynthesis of secondary metabolites and facilitating green biomanufacturing.

[0003] As an important strain for producing natural pesticides, Streptomyces has a rich resource of transport proteins in its genome. Transport proteins account for 12.1% of the protein group of Streptomyces coelicolor, 13.7% of the protein group of Streptomyces griseus, and 12.9% of the protein group of 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 Bulletin, 2020, 47 (11): 3689-98.). Therefore, transporter engineering based on the discovery and engineering of transport systems is an effective strategy for constructing high-yield industrial production strains. Summary of the invention

[0004] In order to increase the yield of natural products of Streptomyces, the present invention screens transport proteins related to yield and production timing based on the analysis of transcriptome data of Streptomyces caniferus NEAU6, and obtains the candidate transport protein T6417 through functional annotation analysis; then, the expression of T6417 is adapted using different promoters, and it is found that overexpressing the transport protein T6417 in Streptomyces caniferus, Streptomyces glacialis or Streptomyces avermitilis can increase the yield of natural products.

[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 a transport protein T6417, the amino acid sequence of the transport protein T6417 is shown in SEQ ID NO.1.

[0007] The second object of the present invention is to provide a gene encoding the above-mentioned transport protein T6417, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The third object of the present invention is to provide a recombinant vector containing the above gene.

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

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

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

[0012] The seventh object of the present invention is to provide a recombinant bacterium, wherein the recombinant bacterium 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 bacteria, which comprises the following steps: constructing a recombinant plasmid containing a transport protein T6417 encoding gene, transforming the recombinant plasmid into Escherichia coli, and introducing the recombinant plasmid into the starting Streptomyces by conjugation transfer.

[0014] In one embodiment of the present invention, 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 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 fusca, Streptomyces bingchengensis or Streptomyces avermitilis.

[0016] Beneficial effects of the present invention:

[0017] The present invention is based on the analysis of the transcriptome data of Streptomyces caniferus NEAU6, screens transport proteins related to yield and production timing, obtains the candidate transport protein T6417 through functional annotation analysis; then uses different promoters (PermE* , P hrdB , P 1140 , P 7518 and P 7518-ant ) adapted the expression of T6417 and found that overexpression of the transporter T6417 in Streptomyces glaucoma, Streptomyces glaucoma or Streptomyces avermitilis could increase the yield of natural products. Among them, the recombinant strain N6-P obtained by overexpressing the transporter T6417 in Streptomyces glaucoma NEAU6 was used as the starting strain. ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417 reached 665mg / L, 658mg / L, 705mg / L, 673mg / L, and 903mg / L, respectively. Compared with the starting strain NEAU6, the oryzanol production of the five recombinant strains increased by 27.2%, 22.4%, 31.3%, 25.3%, and 77.4%, respectively. Taking Bingcheng Streptomyces BC-101-4 as the starting strain, the recombinant strain BC-P obtained by overexpressing the transporter protein T6417 7518-ant -6417 produced milbemycin A4 of 1259 mg / L, which was 13.2% higher than that of the starting strain BC-101-4 of Streptomyces avermitilis. The recombinant strain S0-P obtained by overexpressing the transporter protein T6417 was 7518-ant The avermectin yield of -6417 reached 3g / L, and the avermectin B1a yield was increased by 50% compared with the starting strain Streptomyces avermitilis S0. The present invention provides a valuable biosynthetic element for efficient biomanufacturing of Streptomyces. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 The result diagram of transmembrane prediction for transporter T6417;

[0020] Figure 3 Schematic diagram of the construction of the recombinant vector; wherein, Figure 3 A in the figure is the recombinant vector P ermE* -6417, P hrdB -6417, P 1140 -6417 and P 7518 -6417 construction diagram, Figure 3 B in the figure is the recombinant vector P 7518-ant -6417 construction diagram;

[0021] Figure 4 Streptomyces NEAU6 and recombinant strain N6-P ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417 test result of oryzanol production;

[0022] Figure 5 Streptomyces BC-101-4 and recombinant strain BC-P 7518-ant -6417 Milbemycin production test results;

[0023] Figure 6 Streptomyces avermitilis S0 and recombinant strain S0-P 7518-ant -6417 Avermectin production test results. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Table 1 Strain information involved in the examples

[0028]

[0029] Table 2 Plasmid information involved in the embodiment

[0030]

[0031] Table 3 Primer information involved in the examples

[0032]

[0033]

[0034] Note: ermE* -F / R is used to amplify P ermE* Fragment, P hrdB -F / R is used to amplify P hrdB Fragment, P 1140 -F / R is used to amplify P 1140 Fragment, P 7518 -F / R is used to amplify P 7518 Fragment, ant-F / R for amplification of 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 were used to amplify the scn6417 fragment.

[0035] The strains used in the present invention are from the following sources:

[0036] Streptomyces caniferus NEAU6 is recorded in the Chinese patent application number 201910189570.2, with the invention name of a strain of Streptomyces and its application, and the public can obtain it through the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0037] 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.

[0038] 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.

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

[0040] The solid culture medium required for the growth and fermentation of Streptomyces fusca is YMS solid culture medium, which consists of 4 g / L yeast extract powder, 10 g / L malt extract powder, 4 g / L soluble starch, 20 g / L agar, the balance is water, and the pH is 7.4.

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

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

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

[0044] The composition of the Bingcheng Streptomyces 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.

[0045] The composition of the fermentation medium of Streptomyces bingchengensis: 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.

[0046] The solid medium required for the growth and fermentation of Streptomyces avermitilis is GYM solid medium, which has the following composition: 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, 20g / L agar powder, and the balance is water.

[0047] The composition of the Streptomyces avermitilis 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.

[0048] The composition of the fermentation medium of Streptomyces avermitilis: 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.

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

[0050] DNA polymerase 2×SuperNova PCR Mix (Dye) was purchased from GenStar, Gibson assembly enzyme Exnase MultiS was purchased from Vazyme, restriction endonucleases were purchased from Takara and NEB, agarose gel recovery kit was purchased from OMEGA, plasmid extraction kit was purchased from Axygen, RNA extraction kit was purchased from Beijing Kangwei Technology Co., Ltd., reagents used for RNA purification, reverse transcription and fluorescence quantification were purchased from Thermo Fisher, and chromatographic grade methanol and acetonitrile reagents used for HPLC were all from MREDA.

[0051] The polymerase chain reaction (PCR), agarose gel recovery, Gibson assembly and other operations involved in the present invention are all performed according to the instructions of the reagents and the kit.

[0052] The amino acid sequence of the efflux transporter T6417 of the present invention is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene scn6417 is shown in SEQ ID NO.2. ermE* , P hrdB , P 1140 , P 7518 , and the nucleic acid sequences 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] 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 15 min, and the upper avermectin extract is drawn through a 0.22 μM organic filter membrane for 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] Milbemycin detection method: 0.5 mL of the fermentation broth of Streptomyces bingchengensis was mixed with 1.5 mL of ethanol, and 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, and the upper milbemycin extract was drawn 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: 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.

[0070] The detection method of oryzanin is as follows: 0.4 mL of fermentation broth of Streptomyces fusca was collected, 1.60 mL of ethanol was added, and the oryzanin in the fermentation broth was extracted by ultrasonic treatment for 60 min, and the oryzanin in the fermentation broth was extracted, and the oryzanin in the fermentation broth was centrifuged at 12000 rpm for 10 min. The supernatant was passed through a 0.22 μM organic filter membrane and then liquid phase detection was performed, the detection instrument was Agilent 1260 high performance liquid chromatography (HPLC), the chromatographic column was C18-aq column (ZorbaxSB-Aq, 4.6 mm×250 mm, 5 μm), acetonitrile with a volume fraction of 11% and ultrapure water with a volume fraction of 89% were used as mobile phases, the flow rate was 0.8 mL / min, the detection wavelength was 260 nm, and the column temperature was 28° C.

[0071] Example 1: Acquisition of transporter protein T6417

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

[0073] The amino acid sequence of the transporter protein 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 DeepTMHMM1.0 (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 were as follows: Figure 1 and Figure 2 As shown. Figure 1 It can be seen that the N-terminus and C-terminus of T6417 are on the same side, and the sequence structure shows multiple transmembrane helices repeated, showing a unique "MFS Fold" in the secondary structure domain. Therefore, it is speculated that T6417 is an MFS superfamily transport protein (Deng Dong, Yan Ning. Structural basis and transport mechanism of MFS superfamily transport proteins [J]. Science Bulletin, 2015, 60: 720-728.). In addition, Figure 2 The transmembrane domain prediction results also showed that T6417 has the structural characteristics of multiple transmembrane repeats.

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

[0075] In the life cycle of Streptomyces, the biosynthesis of natural products generally occurs in the late growth period, so the present invention has adapted the expression of T6417 by using different promoters. Candidate promoters include constitutive promoters P ermE* and P hrdB , sequential promoter P 1140 , P 7518 and P 7518-ant Among them, the sequential promoter P 1140 The promoter with the highest expression intensity in the GV biosynthetic gene cluster of Streptomyces NEAU6 is P 7518 The promoter was obtained by screening the whole genome of Streptomyces NEAU6, with the specific screening criteria of FPKMD3 < 300 and FPKMD5 > 500. Then, the promoter P was obtained by heat map clustering. 7518 . Sequential promoter P 75118-ant P 7518 The recombinant promoter equipped with a gene amplifier can amplify the promoter P while expressing in time. 7518 intensity of expression.

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

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

[0078] The recombinant plasmid P successfully verified in Example 2 ermE* -6417, P hrdB -6417, P 1140 -6417, P 7518 -6417, P 7518-ant The plasmids were introduced into Escherichia coli ET12567 (pUZ8002) by heat shock transformation, and the obtained Escherichia coli were inoculated in LB liquid medium containing apramycin, kanamycin, and chloramphenicol (final concentrations of 100 μg / mL) for culture. Then, the plasmids were introduced into Streptomyces 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 NEAU6, T6417 was found to be a putative macrolide efflux protein. Therefore, the milbemycin-producing strain Streptomyces BC-101-4 and the avermectin-producing strain Streptomyces avermitilis S0 were selected as examples to verify the effect of T6417 on the yield of polyketide natural products. Based on the previous data, the recombinant plasmid P 7518-ant -6417 Perform subsequent verification.

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

[0081] The specific operation method of the above-mentioned joint transfer is referred to the following documents:

[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 of two LacI family regulators, 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. Previously unidentified sugar transporter for engineering of high-yield Streptomyces[J].AppliedMicrobiology andBiotechnology, 2024,108(1).

[0085] Example 4: Application of the recombinant strain of Streptomyces NEAU6 in increasing the yield of oryzanin

[0086] The five recombinant strains N6-P ermE* -6417, N6-P hrdB -6417, N6-P 1140 -6417, N6-P 7518 -6417, N6-P 7518-ant -6417 was inoculated on the spore-producing medium YMS and cultured for 7 days, then an appropriate amount of spores were scraped with an inoculation spatula and inoculated into the seed medium of Streptomyces fusca, and cultured at 28°C and 250rpm for 24h. Then, the seed liquid was inoculated with 5% inoculum and inoculated into the fermentation medium of Streptomyces fusca, and cultured at 28°C and 250rpm for 7 days, and the fermentation liquid was collected and the yield of oryzanin was detected.

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

[0088] Example 5: Application of the recombinant strain of Streptomyces BC-101-4 in increasing the yield of milbemycin

[0089] The recombinant strain BC-P 7518-ant-6417 was inoculated on the spore-producing medium SKYM, 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 and 250rpm for 46 hours. Then, the seed liquid was drawn with an inoculum amount of 6% and inoculated into the fermentation medium of Streptomyces bingchengensis, and cultured at 28°C and 250rpm for 9 days. The fermentation liquid was collected and the yield of milbemycin was detected.

[0090] The results are as follows Figure 5 As shown, the recombinant strain BC-P 7518-ant -6417 reached 1259 mg / L, which was 13.2% higher than that of the original strain Streptomyces BC-101-4, indicating that the recombinant vector P 7518-ant -6417 is an effective biosynthetic element that can increase the production of natural products in Streptomyces and is of great significance for the synthesis of natural products in microorganisms.

[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 spore-producing medium GYM, cultured at a constant temperature of 28°C for 6 days, and a bacterial sheet of about 1 square centimeter was taken with an inoculation shovel and inoculated into the seed medium of Streptomyces avermitilis, and cultured at 28°C and 250rpm for 40h. Then, the seed liquid was inoculated into the fermentation medium of Streptomyces avermitilis at an inoculation amount of 6%, and cultured at 28°C and 250rpm for 10 days, and the fermentation liquid was collected and the yield of avermectin was detected.

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

[0094] 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. A transport protein T6417, characterized in that The amino acid sequence of the transporter protein T6417 is shown in SEQ ID NO.

1.

2. A gene encoding the transporter T6417 according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that: The recombinant vector contains the gene according to claim 2.

4. Use of the transporter T6417 according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in constructing a recombinant Streptomyces caniferus that produces oryzanin.

5. Use of the transporter protein T6417 according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in constructing a recombinant Streptomyces bingchenggensis that produces milbemycin.

6. Use of the transporter T6417 according to claim 1, the gene according to claim 2 or the recombinant vector according to claim 3 in constructing a recombinant Streptomyces avermitilis producing avermectin.

7. A recombinant bacterium, characterized in that: The recombinant bacteria contains the gene according to claim 2 or the recombinant vector according to claim 3.

8. The method for constructing the recombinant bacteria according to claim 7, characterized in that: The method comprises the following steps: constructing a recombinant plasmid containing a transport protein T6417 coding gene, transforming the recombinant plasmid into Escherichia coli, and introducing the recombinant plasmid into a starting Streptomyces through conjugation transfer.

9. The construction method according to claim 8, 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 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.

10. The construction method according to claim 8, characterized in that: The starting Streptomyces is Streptomyces fusca, Streptomyces bingchengensis or Streptomyces avermitilis.

Images