A sbhR gene-deficient Streptomyces bingchengensis and its preparation method and application

Abstract

A sort of sbhR The invention discloses a gene-deficient Streptomyces bingchengensis and a preparation method and application thereof, which belong to the field of genetic engineering technology. In order to improve the yield of nanchangmycin produced by Streptomyces bingchengensis, the invention analyzes the whole genome and transcriptome of Streptomyces bingchengensis and finds a gene with a nucleotide sequence as shown in SEQ ID NO.1. sbhR The gene encodes a transcriptional regulator of the DeoR family, which is expressed in Streptomyces glacialis ( Streptomyces bingchenggensis ) BC04 knockout sbhR Gene, constructed sbhR The gene-deficient recombinant strain RsbhR of Streptomyces bingchengensis was found to have an approximately 5.95-fold increase in the yield of nanchangmycin produced by fermentation compared to the starting strain BC04, and can be used to increase the yield of nanchangmycin.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and specifically relates to a sbhR Gene-deficient Streptomyces glacialis and its preparation method and application. Background Art

[0002] Nanchangmycin, also known as Lieshenmycin abroad, is an antibacterial substance mainly used in agricultural research. It is an acidic ester-soluble polyether antibiotic. Nanchangmycin is mainly used to inhibit Gram-positive bacteria, has strong anticoccidial activity, and can also effectively inhibit Zika virus (ZIKV) infection and dengue virus (DENV) infection.

[0003] Streptomyces icebergii Streptomyces bingchenggensis )BC04 is an industrial milbemycin producer. Under the laboratory fermentation conditions for the main production of milbemycin, this Streptomyces also produces a polyether antibiotic: nanchangmycin. In Streptomyces, antibiotic synthesis is tightly regulated by different levels of regulatory factors, including intracluster regulatory factors, pleiotropic regulatory factors, and global regulatory factors. DeoR family transcriptional regulatory proteins are widely present in Streptomyces. However, so far, the function and regulatory mechanism of DeoR regulatory proteins in Streptomyces have been less studied. Therefore, mining novel transcription factors that affect nanchangmycin synthesis from Streptomyces and analyzing the corresponding regulatory mechanisms will provide important high-yield breeding gene resources for the construction of high-yield nanchangmycin strains. Summary of the invention

[0004] In order to improve the yield of nanchangmycin produced by Streptomyces bingchengensis, the present invention analyzes the genome and transcriptome data of Streptomyces bingchengensis and finds a DeoR family transcriptional regulatory gene sbhR The nucleotide sequence of the regulatory gene is shown in SEQ ID NO.1. sbhR Gene, constructed sbhR The gene-deficient recombinant strain RsbhR of Streptomyces bingchengensis was found through fermentation experiments. sbhR The negative regulatory gene for nanchangmycin synthesis was knocked out using Streptomyces BC04 as the starting strain. sbhR Genetically constructed recombinant bacteria can be used to increase the production of Nanchangmycin.

[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 sbhR A gene-deficient recombinant strain of Streptomyces bingchengensis, characterized in that the recombinant strain of Streptomyces bingchengensis is based on Streptomyces bingchengensis (Streptomyces bingchenggensis ) BC04 is the starting strain, and knockout sbhR obtained by gene sbhR The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] In one embodiment of the present invention, the gene deletion refers to gene inactivation, i.e., one or more base point mutations, deletions, insertions or rearrangements in the gene nucleotide sequence. "Inactivation" includes partial inactivation and complete inactivation, which means that the gene function is partially or completely lost, the protein encoded by it cannot be produced and expressed, or the protein expression level is reduced or eliminated, or the relevant biological activity of the expressed protein is reduced or disappeared, for example, the gene cannot be transcribed or the transcribed RNA cannot be translated into a protein with corresponding activity, or the amount of the protein produced or its activity is reduced or disappeared compared with the amount or activity of the protein translated by the gene that has not undergone the inactivation operation.

[0008] Those skilled in the art will appreciate that any known gene inactivation method suitable for Streptomyces can be used to perform gene inactivation in the present invention, including but not limited to gene replacement, gene knockout, insertion inactivation, frameshift mutation, site-directed mutagenesis, partial gene deletion, gene silencing, RNAi, antisense inhibition, etc. For gene inactivation by the above methods, reference can be made to textbooks, technical manuals and references known in the art (e.g., KIESER, T. et al. Practical Streptomyces genetics: A Laboratory Manual. [J]. 2000; Li, T. et al.CRISPR-Cpf1-Assisted Multiplex Genome Editing and Transcriptional Repressionin Streptomyces [J]. Appl Environ Microbiol, 2018, 84).

[0009] In one embodiment of the present invention, the starting strain is Streptomyces BC04.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned recombinant strain of Streptomyces glacialis, the preparation method comprising the following steps:

[0011] 1) Construction of target gene knockout plasmid: Using plasmid pSETddCpf1 as template and RsbhR-F and RsbhR-R as primers, PCR amplified the target gene knockout plasmid. sbhRThe sgRNA fragment of the gene was connected to the vector pSETddCpf1 to obtain the knockout plasmid pSETddCpf1 / sbhR;

[0012] 2) The knockout plasmid pSETddCpf1 / sbhR obtained in step 1) was introduced into Escherichia coli, and then transferred into Streptomyces bingchengensis as the starting strain by inter-genus conjugation to obtain sbhR Gene-deleted Streptomyces icebergii.

[0013] In one embodiment of the present invention, the nucleotide sequences of RsbhR-F and RsbhR-R in step 1) are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0014] In one embodiment of the present invention, step 1) is to combine the sgRNA fragment with Spe I and Nde The vector pSETddCpf1 treated with double enzymes was assembled using the ClonExpress MultiS kit to obtain the knockout plasmid.

[0015] In one embodiment of the present invention, the Escherichia coli in step 2) is ET12567 / pUZ8002.

[0016] The third object of the present invention is to provide an application of the above-mentioned Streptomyces bingchengensis recombinant strain in the production of Nanchangmycin, wherein the application is to produce Nanchangmycin after fermentation and cultivation of the Streptomyces bingchengensis recombinant strain.

[0017] In one embodiment of the present invention, the fermentation culture refers to inoculating the recombinant strain of Streptomyces glacialis into a seed culture medium and a fermentation culture medium in sequence for culture.

[0018] In one embodiment of the present invention, the fermentation medium is composed of: 20 g / L soybean cake powder, 80 g / L sucrose, 1 g / L skimmed milk powder, 0.1 g / L FeSO4·7H2O, 1 g / L K2HPO4, 3 g / L CaCO3, and the balance is water, with a pH of 7.0.

[0019] In one embodiment of the present invention, the composition of the seed culture medium is: sucrose 10 g / L, skimmed milk powder 1 g / L, yeast extract powder 5 g / L, bacterial peptone 3.5 g / L, K2HPO4 0.5 g / L, the balance is water, and the pH is 7.2.

[0020] Beneficial effects of the present invention:

[0021] The present invention analyzes the whole genome and transcriptome of Streptomyces glacialis and finds a DeoR family transcriptional regulatory factor SbhR with a transcription level (FPKM) greater than 1000. The transcriptional regulatory factor has a nucleotide sequence as shown in SEQ ID NO.1. sbhR Gene encoding. Streptomyces bingchenggensis ) BC04 knockout sbhR Gene, constructed sbhR The gene-deficient recombinant strain RsbhR of Streptomyces bingchengensis was found in fermentation experiments. The yield of nanchangmycin produced by strain RsbhR was increased by about 5.95 times (from 1098.4 mg / L to 7635.5 mg / L) compared with the starting strain BC04, indicating that SbhR negatively regulates the synthesis of nanchangmycin. sbhR The gene-deficient recombinant strain of Streptomyces bingchengensis can be used to increase the yield of nanchangmycin produced by Streptomyces bingchengensis fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram for the construction of the recombinant plasmid pSETddCpf1 / sbhR;

[0023] Figure 2 This is a graph showing the detection results of the nanchangmycin content in the fermentation broth of the recombinant strain RsbhR and the original strain BC04. 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 ClonExpress MultiS kit used in the present invention was purchased from Novozymes.

[0027] The solid culture medium, seed culture medium, fermentation medium formula and specific detection methods corresponding to the secondary metabolites used in the present invention are all recorded in the following literature: ZHANG, Y. et al. Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis [J]. Microb Cell Fact, 2016 (15): 152.

[0028] The culture medium used in the present invention and its composition are as follows:

[0029] The composition of SKYM medium is as follows:

[0030] Sucrose 4 g / L, skimmed milk powder 1 g / L, yeast extract powder 2 g / L, malt extract powder 5 g / L, agar powder 20 g / L, the balance is water, pH 7.2.

[0031] The composition of the Streptomyces icebergii seed culture medium is as follows:

[0032] Sucrose 10 g / L, skimmed milk powder 1 g / L, yeast extract powder 5 g / L, bacterial peptone 3.5 g / L, K2HPO4 0.5 g / L, the balance is water, pH 7.2.

[0033] The composition of the fermentation medium of Streptomyces bingchengensis is as follows:

[0034] Soybean cake powder 20 g / L, sucrose 80 g / L, skimmed milk powder 1 g / L, FeSO4·7H2O 0.1 g / L, K2HPO4 1 g / L, CaCO3 3 g / L, the balance is water, pH 7.0.

[0035] The starting strain used in the present invention is:

[0036] Streptomyces icebergii Streptomyces bingchenggensis BC04 is an industrial milbemycin producer, which is described in ZHANG, Y. et al. Characterization of a pathway-specific activator ofmilbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis[J]. Microb Cell Fact, 2016 (15): 152. The above strains are available to the public through the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0037] Definitions and abbreviations in the present invention:

[0038] In the present invention, the starting strain for producing the recombinant strain refers to a Streptomyces strain subjected to the genetic manipulations described in the present invention, such as gene knockout, or a Streptomyces strain with other genetic modifications but not the genetic modification described in the present invention, such as a complemented strain. "Recombinant" as used herein refers to a strain with the desired modification obtained due to intentional human intervention, for example, compared with the corresponding natural (non-recombinant) strain, the recombinant strain does not express or restores the expression of natural gene activity.

[0039] In the present invention, the term "inhibition" means that compared with the starting strain, the recombinant strain is subjected to the genetic manipulation shown in the present invention, so that the recombinant strain cannot produce a protein with corresponding activity, or the amount of the protein produced or the activity of the protein produced is reduced or eliminated compared with the amount or activity of the protein translated from the gene that has not undergone the inactivation operation.

[0040] The plasmid information involved in the following examples is shown in Table 1; the strain information involved is shown in Table 2; and the primer information involved is shown in Table 3.

[0041] Table 1 Plasmid information involved in the examples

[0042]

[0043] Table 2 Information on strains involved in the examples

[0044]

[0045] Table 3 Primer sequence information involved in the examples

[0046]

[0047] Embodiment 1: sbhR Construction of the gene-deleted recombinant strain RsbhR of Streptomyces bingchengensis

[0048] The recombinant strain RsbhR is based on Streptomyces BC04, and the gene sbhR The specific construction method is as follows:

[0049] (1) Construction of target gene suppression vector

[0050] The polymerase chain reaction (PCR) and KOD high-fidelity enzyme system were used to amplify the specific targeting sbhR Gene( sbhR The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the SbhR protein is shown in SEQ ID NO.2) and the PCR reaction system (total volume 50 μL) is as follows: 10×KOD buffer 5 μL, 10×dNTPs 5 μL, Mg 2+ 2 μL, 10 μM RsbhR-F 2 μL, 10μM RsbhR-R 2 μL, DMSO 3 μL, pSETddCpf1 1 μL, ddH2O 29.5 μL, KOD plus 0.5 μL. PCR reaction conditions were as follows: 94°C 4 min, 94°C 1 min, 66°C 30 sec 35 cycles, 68°C 30 sec, 68°C 5 min, and stored at 4°C. Spe I and Nde I double enzyme digestion (the total volume of the double enzyme digestion system is 100 μL, which is composed of the following components: 3 μL of pSETddCpf1 plasmid, 10 μL of enzyme buffer, Spe I enzyme 3 μL, Nde I enzyme 3 μL, ddH2O 81 μL), was assembled using the ClonExpress MultiS kit, introduced into JM109 by transformation, and after plating, clones were picked and cultured in a small test tube containing apramycin (final concentration of 100 μg / mL). The plasmid was extracted with a plasmid extraction kit, and the recombinant plasmid pSETddCpf1 / sbhR (such as Figure 1 ).

[0051] SEQ ID NO.1:

[0052] gtgttcgctgcagaacgtcgccaattgatccttgaaatggtgcgtgcgaacggagcggtatcgctccgcgagctcgcccgcgtcgtccagacctccgaagtgaccgtacggcgggatgtgcgggcgctggaggcagaaggactgctcgaccgccggcacggcggtgcggtgctgccgggcggattcaccagggagtccggcttcccgcagaaatcccatctagcgaccgcggagaagacggccatcgccgatctcgcggccggtctcgtcgacgagggcgaggccgtcgtggtcggcgccgggaccaccacgcaagagctggcccgccggctcgcgcgcgtccctggcctgaccgtggtcaccaactccctgctggtcgcacaggcattggcccatgccaacagggtggaggtcgtcatgaccggcggcaccctgcgcggctccaactacgcgctggtcggcagcggtgccgagcagtccctgcaggggctgcgggtcagccgggcctttctgtccggcagtgggctgaccgccgagcgcggcctgtccacgtccaacatgctgtcggccagcgtcgaccgggcgctggtgcaggcggcggcggaggtggtggtcctcgcggaccacaccaaactcggcaccgacaccatgttccagacggtgcctacggatgtgatcacccgtctggtcacggacgagccgccgccgcatgacgaccgcgccctgaccgagctgcaggccctcgccgaccagggcgtgcagatcgcggtggccgggccgggcgcgggctcgggcccggcgggcggtgagggcggcccggcggcaccgggccgccagcagccccggctcggcggcccaccccccgcccgcggtacggaggaggggctgggggggccgcggcccgccccccccccgcggtag

[0053] SEQ ID NO.2:

[0054] VFAAERRQLILEMVRANGAVSLRELARVVQTSEVTVRRDVRALEAEGLLDRRHGGAVLPGGFTRESGFPQKSHLATAEKTAIADLAAGLVDEGEAVVVGAGTTTQELARRLARVPGLTVVTNSLLVAQALAHANRVEVVMTGGTLRGSNYALV GSGAEQSLQGLRVSRAFLGSGLTAERGLSTSNMLSASVDRALVQAAAEVVVLADHTKLGTDTMFQTVPTDVITRLVTDEPPPHDDRALTELQALADQGVQIAVAGPGAGSGPAGGEGGPAAPGRQQPRLGGPPPARGTEEGLGGPRPAPPPR*

[0055] (2) Construction of recombinant strains

[0056] The recombinant plasmid pSETddCpf1 / sbhR was transferred into ET12567 / pUZ8002, and then introduced into Streptomyces BC04 by conjugation transfer. The conjugate was then picked onto SKYM medium containing apramycin (final concentration 8 μg / mL) and nalidixic acid (final concentration 25 μg / mL), cultured at 28°C for 9 days, and then the strain was subcultured onto new SKYM medium containing apramycin (final concentration 8 μg / mL), and cultured at 28°C for 9 days before the cells were collected.

[0057] Example 2: Application of the recombinant strain RsbhR of Streptomyces bingchengensis in the fermentation production of Nanchangmycin

[0058] The method for producing nanchangmycin using the recombinant strain RsbhR is as follows: the recombinant strain RsbhR is inoculated into SKYM solid culture medium, cultured at a constant temperature of 28°C for 9 days, spores of about 1 square centimeter are scraped, inoculated into Bingcheng Streptomyces seed culture medium, and cultured at 28°C, 250 rpm for 46 hours; then, the obtained seed liquid is inoculated into Bingcheng Streptomyces fermentation medium at an inoculation rate of 6%, and cultured at 28°C, 250 rpm for 9 days.

[0059] HPLC detection verifies the yield change of Nanchangmycin produced by fermentation of recombinant strain RsbhR:

[0060] The recombinant strain RsbhR and the starting strain BC04 were inoculated on SKYM solid medium, cultured at a constant temperature of 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, 250rpm, for 46 hours. Then, the obtained seed liquid was inoculated into the fermentation medium of Streptomyces bingchengensis at an inoculation rate of 6%, and cultured at 28°C, 250 rpm, for 9 days to obtain the fermentation broth of strains BC04 and RsbhR, respectively.

[0061] The detection method of nanchangmycin production is as follows: centrifuge 1 mL of fermentation culture, discard the supernatant, resuspend the bacteria with 1 mL of methanol, and rotate overnight for extraction. Centrifuge at 12000 rpm for 10 min, take the supernatant, filter through a 0.22 μm organic filter membrane, and perform HPLC detection. Mobile phase A is deionized water containing 0.2% formic acid, and mobile phase B is acetonitrile. Liquid phase conditions: mobile phase A: mobile phase B = 1:9 (volume ratio), flow rate 1 mL / min, column temperature 28°C, detection wavelength 230 nm.

[0062] HPLC test results are as follows Figure 2 As shown in the figure, it can be seen that the nanchangmycin production of the recombinant strain RsbhR (7635.5 mg / L) is about 5.95 times higher than that of the starting strain BC04 (1098.4 mg / L), indicating that SbhR negatively regulates the synthesis of nanchangmycin. sbhR The gene-deficient recombinant strain of Streptomyces bingchengensis can be used to increase the yield of nanchangmycin produced by Streptomyces bingchengensis fermentation.

[0063] 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 oeLh A gene-deficient recombinant strain of Streptomyces bingchengensis, characterized in that: The recombinant strain of Streptomyces bingchengensis is based on Streptomyces bingchengensis ( Streptomyces bingchenggensis ) is the starting strain, and knockout oeLh obtained by gene oeLh The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The recombinant strain of Streptomyces glacialis according to claim 1, characterized in that The starting strain is Streptomyces BC04.

3. The method for preparing the recombinant strain of Streptomyces glacialis according to any one of claims 1 or 2, characterized in that: The following steps are involved: 1) Using plasmid pSETddCpf1 as template and RsbhR-F and RsbhR-R as primers, PCR amplified the specific targeting oeLh The sgRNA fragment of the gene was connected to the vector pSETddCpf1 to obtain the knockout plasmid pSETddCpf1 / sbhR; 2) The knockout plasmid pSETddCpf1 / sbhR obtained in step 1) was introduced into Escherichia coli, and then transferred into Streptomyces bingchengensis as the starting strain by inter-genus conjugation to obtain oeLh Gene-deleted Streptomyces icebergii.

4. The preparation method according to claim 3, characterized in that: Step 1) The nucleotide sequences of RsbhR-F and RsbhR-R are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

5. The preparation method according to claim 3, characterized in that: The step 1) is to combine the sgRNA fragment with Spe I and N The vector pSETddCpf1 treated with double enzymes was assembled using the ClonExpress MultiS kit to obtain the knockout plasmid.

6. The preparation method according to claim 3, characterized in that: In step 2), the Escherichia coli is ET12567 / pUZ8002.

7. Use of the recombinant strain of Streptomyces bingchengensis according to any one of claims 1 or 2 in the production of nanchangmycin, characterized in that: The application is to produce Nanchangmycin by fermenting and culturing the recombinant strain of Streptomyces bingchengensis.

8. The use according to claim 7, characterized in that: The fermentation culture refers to inoculating the recombinant strain of Streptomyces bingchengensis into a seed culture medium and a fermentation culture medium in sequence for culture.

9. The use according to claim 8, characterized in that: The fermentation medium is composed of: 20 g / L soybean cake powder, 80 g / L sucrose, 1 g / L skimmed milk powder, 0.1 g / L FeSO4·7H2O, 1 g / L K2HPO4, 3 g / L CaCO3, and the balance is water, with a pH of 7.

0.

10. The use according to claim 8, characterized in that: The seed culture medium is composed of: 10 g / L sucrose, 1 g / L skimmed milk powder, 5 g / L yeast extract powder, 3.5 g / L bacterial peptone, 0.5 g / L K2HPO4, and the balance is water, with a pH of 7.2.

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