阅读说明：本技术 一种玫瑰孢链霉菌中提高癸酸利用率及耐受度的调控基因及应用 (Regulatory gene for improving utilization rate and tolerance of capric acid in streptomyces roseosporus and application of regulatory gene ) 是由 吴杰群 吴金荣 方丽纳 张薇 杨永梅 范萍 沈建宇 徐金勇 方一民 夏迷妮 储消 于 2020-05-26 设计创作，主要内容包括：本发明公开了一种玫瑰孢链霉菌生产达托霉素的调控基因,其核苷酸序列为SEQ ID NO：1所示,或与SEQ ID NO：1所示核苷酸编码相同蛋白的核苷酸序列。本发明通过使用全局调控因子CRP,解决了传统链霉菌育种的高工作量,高盲目性的问题,得到了利用基因工程培育的达托霉素高产的菌株,应用前景广阔；利用该方法对出发菌玫瑰孢链霉菌进行改造,意外发现CRP基因能提高玫瑰孢链霉菌在发酵过程中对癸酸的利用率,以及提高其对癸酸的耐受度。(The invention discloses a regulatory gene for producing daptomycin by streptomyces roseosporus, which has a nucleotide sequence of SEQ ID NO: 1, or a sequence similar to that shown in SEQ ID NO: 1 encodes the same protein. According to the invention, by using the global regulatory factor CRP, the problems of high workload and high blindness of traditional streptomycete breeding are solved, and the high-yield daptomycin strain cultured by using genetic engineering is obtained, so that the application prospect is wide; the method is used for modifying the streptomyces roseosporus serving as the starting bacterium, and the CRP gene is unexpectedly found to improve the utilization rate of the streptomyces roseosporus to the capric acid in the fermentation process and improve the tolerance of the streptomyces roseosporus to the capric acid.)

1. The CRP protein capable of improving the yield of daptomycin in Streptomyces roseosporus is characterized in that the protein sequence is shown in SEQ ID No. 3.

2. The CRP protein according to claim 1, wherein said protein is expressible in streptomyces roseosporus.

3. The CRP protein of claim 2, wherein the coding nucleotide sequence of said protein is set forth in SEQ ID No. 1.

4. A nucleotide fragment encoding the CRP protein of claims 1-3, wherein the nucleotide sequence is set forth in SEQ ID No. 1.

5. A recombinant plasmid is characterized in that a protein shown as SEQ ID NO.3 can be expressed in Streptomyces roseosporus, and preferably, a coding gene of the protein is shown as SEQ ID NO. 1.

6. The recombinant plasmid according to claim 5, wherein the backbone of the plasmid comprises a shuttle plasmid into which a strong promoter erme can be inserted, preferably pKC1139, pEST152, pOJ260 or pSOK804, more preferably pSET 152.

7. The recombinant plasmid according to claim 6, wherein the nucleotide sequence of the plasmid is represented by SEQ ID No. 2.

8. A regulatory gene is characterized by being capable of improving the tolerance and the utilization rate of streptomyces roseosporus to capric acid.

9. The regulatory gene of claim 9, wherein the nucleotide sequence of the regulatory gene is represented by SEQ ID No. 1.

10. Use of a CRP protein according to any one of claims 1 to 3 or a nucleotide fragment according to claim 4 or a recombinant plasmid according to any one of claims 5 to 7 or a regulatory gene according to any one of claims 8 to 9 for increasing the production of daptomycin in streptomyces roseosporus.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a regulatory gene for improving the utilization rate and tolerance of decanoic acid in streptomyces roseosporus, application thereof and a method for obtaining streptomyces with high antibiotic yield.

Background

Daptomycin is a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus and interacts with cell membranes in a calcium ion dependent manner and exerts bactericidal activity. In 12 months 2010, the FDA in the united states approved a new dosing regimen of tobramycin (daptomycin) injection from Cubist pharmaceutical co. Cubicin was approved in the earliest united states in 2003 for 1-30 min intravenous infusion administration a day for the treatment of complicated skin and skin tissue infections caused by certain gram-positive bacteria, including methicillin-resistant staphylococcus aureus, and in 2006 a new indication for the treatment of bacteremia caused by methicillin-sensitive and resistant staphylococcus aureus, including right heart infective endocarditis. The general administration of food and drug administration in China approved daptomycin for injection produced by Hangzhou China, China east China pharmaceutical Co., Ltd, Zhejiang Hazheng pharmaceutical industry Co., Ltd and Jiangsu Hengrui pharmaceutical Co., Ltd in 2015 and 2016, respectively. Multiple studies show that the drug resistance ratio of daptomycin is not obviously improved for more than 10 years, and the unique advantages of daptomycin are revealed.

Daptomycin is a microbial secondary metabolite, has a complex structure and very low yield, and is an important strategy for improving the yield of daptomycin and reducing the production cost through genetic engineering modification.

Capric acid is an exogenous precursor in daptomycin synthesis, and can change the secondary metabolism direction of thalli. Capric acid is essential in daptomycin production, but excess capric acid can be toxic to S.roseosporus; conversely, if the concentration of decanoic acid is too low, the precondition supply is insufficient and the titer of the product in the fermentation broth is reduced. Cyclic adenosine receptor protein (CRP) is a conserved metabolic regulator widely found in bacteria, both gram-negative and gram-positive, but not in bacillus and other firmicutes. CRP has been most widely studied in E.coli. In E.coli, it mediates the carbon catabolism inhibition process together with the allosteric effector cAMP. Among actinomycetes, including Streptomyces, CRP also has an important Global regulatory role, and numerous studies have shown that CRP Is directly involved in the regulation of various antibiotics in Streptomyces coelicolor, indicating that it can influence the flux of precursors into secondary metabolism and plays a role in primary and secondary metabolic processes (Gao C, Hindra, Mulder D, et al. CRP Is a Global Regulator of biological Production in Streptomyces [ J ]. mBio,2012,3 (6)). The nucleotide consistency of CRP of daptomycin and CRP of streptomyces coelicolor is 89.3%, the amino acid sequence consistency is 94.2%, through phylogenetic tree analysis, CRP widely exists in streptomyces and has high conservation, but the functions of CRP in most streptomyces are different, some of CRP can regulate and control the development of streptomyces spores, some of CRP can only regulate and control secondary metabolites, but CRP genes are not reported to improve the tolerance of streptomyces roseosporus to capric acid.

Disclosure of Invention

The invention provides a regulatory gene for improving the utilization rate and tolerance of decanoic acid in streptomyces roseosporus, and provides application of the gene in preparation of a high-yield daptomycin strain.

The regulatory gene for the utilization rate and tolerance of the capric acid in the streptomyces roseosporus provided by the invention can encode CRP protein.

As a specific embodiment, the amino acid sequence of the CRP protein is shown as SEQ ID NO. 3.

As a specific embodiment, the regulatory gene may be expressed in Streptomyces roseosporus.

As a specific embodiment, the nucleotide sequence of the regulatory gene is shown as SEQ ID NO. 1.

As a specific embodiment, the regulatory gene is obtained by subcloning the Streptomyces roseosporus genome and has the length of 672 base pairs.

As a specific implementation mode, the regulatory gene can improve the tolerance and the utilization rate of streptomyces roseosporus to capric acid, and the nucleotide sequence of the regulatory gene is shown as SEQ ID No. 1.

As a specific embodiment, the regulatory gene is a gene with the highest similarity found in the genome sequence of Streptomyces roseosporus by the homology analysis of CRP gene of Streptomyces coelicolor, the gene is named as CDS-7, and the annotation is classified as CRP/Fnr family transcriptionality regulator, but no related literature and research report the function of the regulatory gene in daptomycin, and the regulatory gene is determined to be a CRP homologous protein by further phylogenetic tree and amino acid sequence motif analysis.

The invention also provides a recombinant plasmid which can express the protein of SEQ ID NO.3 in streptomyces roseosporus, and preferably, the coding gene of the protein is SEQ ID NO. 1.

In a specific embodiment, the plasmid backbone of the recombinant plasmid comprises a shuttle plasmid, preferably pKC1139, pEST152, pOJ260 or pSOK804, into which a strong promoter erme can be inserted.

In a specific embodiment, the nucleotide sequence of the recombinant plasmid with pSET152 as the framework is SEQ ID NO. 2.

The invention provides a method for obtaining streptomyces with high antibiotic yield by using a regulatory gene CRP for enhancing the utilization rate of capric acid and improving the tolerance to capric acid in streptomyces roseosporus, which comprises the following steps:

(a) through the homology analysis of CRP genes of streptomyces coelicolor, finding out the gene with the highest similarity in the genome sequence of streptomyces roseosporus, and determining the gene as CRP homologous protein through further phylogenetic tree and amino acid sequence motif analysis;

(b) cloning a target regulatory gene CRP fragment from streptomyces roseosporus by using PCR, and cloning the fragment onto an expression vector to obtain a recombinant plasmid containing the regulatory gene CRP;

(c) transferring the recombinant plasmid containing the regulatory gene CRP into streptomyces roseosporus by a conjugative transfer method;

(d) obtaining an antibiotic producing strain recombinant strain containing a regulatory gene CRP by antibiotic screening;

(e) and (d) performing antibiotic determination on the recombinant bacteria obtained in the step (d) to obtain the streptomyces roseosporus strain with high antibiotic yield.

As a specific embodiment, the plasmid backbone of the expression vector of step (b) comprises a shuttle plasmid, preferably pKC1139, pEST152, pOJ260 or pSOK804, into which a strong promoter erme can be inserted.

As a specific embodiment, the Streptomyces roseosporus in step (c) is Streptomyces roseosporus NO.CGMCC 4.7231.

As a specific embodiment, the Streptomyces roseosporus described in step (e) has been submitted to the general microbiological culture Collection center of China general microbiological culture Collection management Committee in 2019, 7 and 26 months, with the collection addresses: china, Beijing, institute of microbiology, China academy of sciences, with a accession number of CGMCC No. 18297.

The preservation information of the recombinant strain of the invention is as follows:

preservation time: 26 months 7 in 2019;

the name of the depository: china general microbiological culture Collection center;

the preservation number is: CGMCC No. 18297;

the address of the depository: xilu No.1 Hospital No.3, Beijing, Chaoyang, North;

and (3) classification and naming: streptomyces roseosporus (Streptomyces roseosporus).

Therefore, the invention has the following beneficial effects: according to the invention, by using the global regulatory factor CRP, the problems of high workload and high blindness of traditional streptomycete breeding are solved, and the high-yield daptomycin strain cultured by using genetic engineering is obtained, so that the application prospect is wide; the method is used for modifying the streptomyces roseosporus serving as the starting bacterium, and the CRP gene is unexpectedly found to improve the utilization rate of the streptomyces roseosporus to the capric acid in the fermentation process and improve the tolerance of the streptomyces roseosporus to the capric acid.

Drawings

FIG. 1 is a graph showing the comparison of the fermentation yields of Streptomyces roseosporus with high antibiotic production in accordance with the present invention and the fermentation yields of the existing Streptomyces roseosporus after normalization processing at different precursor concentrations in the medium;

FIG. 2 is a physical map of recombinant plasmid pSET152 CRP;

FIG. 3 is an electrophoretogram of pSET152CRP cleavage;

FIG. 4 shows the sequencing of the pSET152CRP plasmid;

FIG. 5 is a comparison graph of normalized daily daptomycin titer of the present invention during fermentation of the producing strain and the constructing strain, with two feeding processes of feeding capric acid in the normal production process daily and feeding capric acid twice the normal amount daily;

FIG. 6 is a comparison graph of the daily decanoic acid utilization rate (titer/cumulative addition of decanoic acid) of the present invention during fermentation process of the present invention with the two feeding processes of feeding decanoic acid in the normal production process daily and feeding twice the normal amount of decanoic acid daily.

Detailed Description

The technical solution of the present invention will be further described with reference to the following embodiments.

In the present invention, all the equipments and materials are commercially available or commonly used in the industry, and the methods in the following examples are conventional in the art unless otherwise specified.

Example 1: construction of pSET152CRP of recombinant plasmid containing CRP gene:

the target gene CRP in Streptomyces roseosporus is obtained by performing Blast on the nucleotide sequence of the homologous gene CRP reported in Streptomyces coelicolor and the genome sequence of Streptomyces roseosporus of NCBI (website: www.ncbi.nlm.nih.gov), and the size of the target gene CRP is 672 bp.

Primers were designed with primer 5.0 as follows:

CRPS:ATTTCTAGAAATACCTGACCGAGCACG；

CRPA:ATTGGATCCATCGCACTGTTTTACCGT。

extracting the total DNA of the streptomyces roseosporus, and amplifying CRP genes to obtain target gene fragments.

Plasmid pSET152 was extracted using a Santerp column plasmid DNA miniprep kit, according to the methods described in the kit instructions. The resulting plasmid DNA solution was stored at-20 ℃ or used for subsequent experiments.

The pSET152 plasmid was treated with restriction enzymes, subjected to DNA agarose gel electrophoresis after digestion, and a desired band of 858bp in size was recovered using a SanPerp column DNA gel recovery kit according to the protocol. The resulting DNA solution was stored at-20 ℃ or used for subsequent experiments.

Connecting the amplified CRP gene with the pSET152 plasmid fragment recovered by enzyme digestion, and carrying out enzyme digestion and connection reaction: 0.03pmol vector, 0.03-0.3pmol fragment, T4 DNA ligase 1200U, 5ul buffer, the remaining volume was made up with distilled water, ligation was performed at 16 ℃ for 16h to obtain ligation product.

Coli competent cells (200 ul) were added to the ligation product, and the contents were mixed by gentle rotation and placed on ice for 30 min. The tube was placed in a circulating water bath pre-warmed to 42 ℃ for 90 s. The tube was quickly transferred to a water bath and the cells were allowed to cool for 1-2 min. 800ul of LB medium pre-warmed at 37 ℃ was added to each tube, which was then transferred to a 37 ℃ water bath for 45 min. Appropriate volumes of transformed competent cells were transferred to LB medium containing the corresponding resistance. Colonies appeared after inverted culture at 37 ℃ for 12-16 h. And selecting colonies for enzyme digestion verification, wherein the verification result is shown in figure 3, selecting colonies which are successfully verified for storage and sequencing verification, and comparing the sequencing result with Snapgene and checking a seq peak map is shown in figure 4.

The size of the recombinant plasmid pSET152CRP is 7033bp, has an ampakia resistance gene and can be screened in escherichia coli and streptomyces roseosporus, int Φ C31 is an integrase gene, attP is an integration site, and p × erme is an erythromycin promoter.

The LB medium formula: 10g of tryptone, 10g of yeast extract and 10g of NaCl, and adding deionized water to 1000ml of water with the pH value of 7.0. Sterilizing at 121 deg.C for 30min, and culturing Escherichia coli. 2% agar powder is added into the solid culture medium.

Example 2: transfer of the recombinant plasmid to S.roseosporus by intergeneric conjugation

Carrying out conjugation transfer on escherichia coli ET12567 containing recombinant plasmid PSET152CRP and Streptomyces roseosporus NO.CGMCCC4.7231, screening by using the apramycin and the nalidixic acid to obtain a conjugator containing PSET152CRP plasmid, and simultaneously using a vector introduced with empty vector PSET152 empty plasmid as a control.

The method for the intergeneric conjugative transfer of Escherichia coli and Streptomyces roseosporus NO.CGMCC4.7231 comprises the following steps:

ET12567(PUZ8002/PSET152) and ET12567(PUZ8002/PSET152CRP) to LB (kanamycin-containing)Chloramphenicol/ampicillin) medium, in a shaker at 37 ℃ and 220rpm overnight. ET12567(PUZ8002/PSET152) and ET12567(PUZ8002/PSET152CRP) were transferred to fresh LB medium (containing kanamycin/chloramphenicol/ampicillin) at a ratio of 1:100, respectively, and cultured on a shaker at 37 ℃ and 220rpm until OD600 reached 0.3-0.4. Cells were resuspended 2 times with the same volume of LB medium and finally in 0.1 volumes of LB medium. While washing the cells, 10-8 spores required for each conjugal transfer were suspended in 500ul 2 XYT medium, heat shocked at 50 ℃ for 10min, and cooled to room temperature. Each 500ul of E.coli solution and S.roseosporus spore solution were mixed well, centrifuged to remove most of the supernatant, and resuspended in the remaining liquid. The mixed bacterial liquid was applied to a suspension containing 10mM MgCl₂The MS solid culture medium is cultured for 16-20h at 29 ℃. 1ml of sterile water containing 0.5mg of nalidixic acid and 1mg of apramycin was applied and evenly covered on the junction-transferred plates. The mixture is further cultured in an incubator at 29 ℃ for 2-3 days, and then the zygote can be observed. A single colony of S.roseosporus containing pSET152CRP was obtained by streaking, and the single colony was inoculated into a 5ml TSB liquid tube containing 75ug nalidixic acid and 250ug apramycin for passaging to verify the genotype of the zygote.

2 XYT culture medium formula:

tryptone 16g, yeast extract 10g, NaCl5g, adding deionized water to 1000ml, adjusting pH to 7.0 with 5N NaOH, and autoclaving at 121 ℃ for 20 min.

MS culture medium formula:

20g of mannitol, 20g of soybean cake powder and 20g of agar, adding deionized water to 1000ml, pH7.2-7.3, sterilizing twice at 115 ℃ for 15min, and adding 1M MgCl when in use₂To a final concentration of 10 mM/L. (1M MgCl)₂： MgCl₂.7H₂O)

Example 3: recombinant bacterium fermentation process

Culturing the constructed Streptomyces roseosporus strain in an R5 slant culture medium at 30 ℃ for 5 days, transferring the strain into a shake flask containing 50ml YEME liquid culture medium, culturing for 25h at 30 ℃ under oscillation (220rpm/min), culturing by taking a one-time supplementary material of capric acid as a fermentation precursor during fermentation until the fermentation is finished, measuring a fermentation unit by HPLC, and selecting a high-yield strain. As shown in the attached figure 1 of the specification, compared with the existing Streptomyces roseosporus (outgrowth), the Streptomyces roseosporus with high antibiotic yield prepared by the invention has higher fermentation yield, namely better daptomycin yield, and the fermentation yield is improved by more than 200%.

TSB liquid medium formula:

17g of tryptone, 3g of soybean peptone, 2.5g of D-glucose, 5g of sodium chloride and 2.5g of dipotassium hydrogen phosphate, adding deionized water to 1000ml, and sterilizing at 121 ℃ for 30 min.

Formula of R5 culture medium:

sucrose 103.0g, K2SO40.25g, MgCl₂·6H₂010.12 g, glucose 10.0g, hydrolyzed casein 0.1g, microelement solution 2.0g, yeast extract 5.0g, TES buffer 57.3ml, KH₂PO₄(0.5％) 10ml，CaCl₂·H₂4ml of O (5M), 150ml of L-proline (20%), 7ml of NaOH (1N), 20.0g of agar, and deionized water to 1000ml, pH 7.2. Sterilizing at 115 deg.C for 30 min.

Trace element solution (per liter): ZnCl₂ 40mg，FeCl₂·6H₂O 200mg，CuCl₂·2H₂O 10mg， MnCl₂·2H₂O 10mg，Na₂B₄O₇·10H₂O 10mg，(NH₄)₆Mo₇O₂₄·4H₂O 10mg

YEME culture medium formula:

1.5g yeast extract, 5g tryptone, 3g malt extract, 10g glucose, 250g sucrose, adding deionized water to 1000ml, and steam sterilizing at 115 deg.C for 15 min.

Example 4: HPLC determination of daptomycin fermentation units

Chromatographic conditions are as follows: a chromatographic column: phenomenex IB-SIL C84.6X 250mm 5um, flow rate: 1.0mL/min, detection wavelength: 223nm, sample size: 25 μ L, column temperature: 25 ℃, gradient elution mobile phase a: 3.4g of ammonium dihydrogen phosphate are weighed out and dissolved in 1000mL of distilled water, the pH is adjusted to 3.1 with phosphoric acid, mobile phase B: and (3) acetonitrile.

Example 5: construction of a Strain tolerance test to precursor concentration

The fermentation process of example 3 was used to compare the tolerance ranges of the starting and construction strains no.cgmcc4.7231 to the concentration of fermentation precursors in the medium in shake flasks. The experimental result is shown in the attached figure 1 of the specification, when the precursor concentration of the construction bacteria in the shake flask is 3% -5%, the yield of daptomycin is highest, and after the precursor concentration exceeds 5%, the yield is obviously reduced; but the precursor concentration with the highest yield of the starting bacteria is 2-3%, and the yield is obviously reduced after 3%. The CRP gene can improve the tolerance of the streptomyces roseosporus to the precursor in the culture medium, and can improve the addition amount of the decanoic acid in a fermentation tank during industrial production to obtain higher yield per unit volume.

Example 6: comparison of pilot-scale fermentation process between spawn-producing strain and constructed strain

According to the pilot fermentation process of daptomycin, the constructed streptomyces roseosporus strain is cultured in a seed tank for 22-26 hours, then transferred into a fermentation tank, cultured for 25 hours at about 30 ℃, decanoic acid is continuously fed as a fermentation precursor during fermentation, and the decanoic acid amount fed into the tank by the construction strain is twice of that of the normal process of the starting strain according to the tolerance of the construction strain to the precursor. In contrast, the starting strain used the same feeding process as the construction strain (i.e., the decanoic acid feed was twice as much as the original process). And culturing until the fermentation is finished, measuring fermentation units by HPLC, and comparing.

The seed tank formula is as follows: 6% of potato starch, 1.5% of glucose, 0.72% of cane molasses, 0.08% of ammonium ferrous sulfate and 0.05% of sodium silicate.

The fermentation tank comprises the following components in percentage by weight: 7.2 percent of potato starch, 1 percent of glucose, 0.72 percent of cane molasses, 1.2 percent of yeast powder, 0.086 percent of ammonium ferrous sulfate and 0.05 percent of foam killer.

As shown in the attached figure 5 of the specification, the titer of the constructed bacteria is 2.5-3 times of the original process of the starting bacteria and is more than 2 times of the same process of the starting bacteria (namely the decanoic acid supplement amount is twice of the original process) on the 7 th day and the 8 th day of fermentation on a tank. The results shown in the attached figure 6 of the specification show that in the fermentation process, the utilization rate of the decanoic acid (calculated by titer/cumulative decanoic acid supplementation) of the constructed bacteria is more than 2 times of that of the original process of the starting bacteria, and meanwhile, the utilization rate of the decanoic acid is obviously improved compared with the same process of the starting bacteria (namely the decanoic acid supplementation is twice of that of the original process). The two figures illustrate that, firstly, the construction bacteria obviously improve the expression quantity of daptomycin on the tank and increase the yield of single fermentation; secondly, the construction bacteria obviously improve the utilization rate of the decanoic acid, so that more daptomycin is generated after the same amount of decanoic acid is added.

It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.