Method for improving fermentation level of clavulanic acid producing strain

Abstract

The invention discloses application of a transcriptional regulation factor SCLAVp0126 in regulation of the fermentation yield of clavulanic acid of a strain, the fermentation yield of clavulanic acid of a mutant strain can be remarkably increased by knocking out the transcriptional regulation factor SCLAVp0126 in a streptomyces clavuliformis genome, and the application has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to use of a transcriptional regulatory factor SCLAV_p0126 in improving the fermentation level of clavulanic acid producing bacteria. Background Art

[0002] Beta-lactam antibiotics include penicillin and its derivatives, cephalosporins, and monoamide ring drugs, and have attracted much attention due to their wide clinical application. However, long-term use may cause pathogens to develop drug resistance, mainly because pathogens can produce beta-lactamase, which destroys the amide ring structure in the antibiotic molecule and thus weakens the efficacy. In order to address the problem of drug resistance, the combined use of beta-lactam antibiotics and beta-lactamase inhibitors has become one of the effective solutions.

[0003] In 1971, Higgens and Kastner isolated clavulanic acid from Streptomyces clavuligerus for the first time (International Journal of Systematic Bacteriology, 1971, 21 (4): 326-330). Subsequent studies have shown that clavulanic acid is an irreversible β-lactamase inhibitor with important medical value. Since its discovery, the development of industrial strains, fermentation process improvements, and finished drug research and development related to it have continued. However, in recent years, there have been few reports on the transformation of this strain to increase the yield of clavulanic acid, mainly by optimizing the fermentation process to increase the yield. Therefore, further developing new strains to improve the fermentation level of clavulanic acid has great economic value. Summary of the invention

[0004] In our ongoing research on the fermentation production of clavulanic acid by Streptomyces clavuligerus, we have noticed that transcription factors play a key role in the regulation of Streptomyces secondary metabolites, which affect the activity of metabolic pathways by regulating gene expression. As an important industrial microorganism, Streptomyces can synthesize a variety of bioactive molecules, such as antibiotics, and the synthesis of these secondary metabolites is usually significantly regulated by specific transcription factors. For example, AdpA, as an important global transcriptional regulator, directly regulates the expression of multiple antibiotic biosynthetic gene clusters. In addition, transcription factors such as the Lrp, TetR and LuxR families play an important role in environmental response and the coupling of primary metabolism and secondary metabolism. By manipulating the expression of transcription factors, it is expected to further increase the production of antibiotics. Based on the biosynthetic pathway of clavulanic acid, a systematic investigation was conducted on the regulatory factors / transcriptional regulatory factors that may regulate clavulanic acid synthesis, and strains were genetically engineered to overexpress and knock out them, respectively. It was found that overexpression of the transcriptional regulatory factor SCLAV_p0126 (GeneBank No. EFG03617.1) did not significantly change the clavulanic acid fermentation level of the engineered bacteria of Streptomyces clavuligerus, but knocking out the gene could significantly increase the clavulanic acid yield, indicating that SCLAV_p0126 negatively regulates the biosynthesis of clavulanic acid. Based on this research finding, the present invention provides the following technical solution.

[0005] The first aspect of the present invention provides the use of the transcriptional regulatory factor SCLAV_p0126 (GeneBank No. EFG03617.1, the nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2) in regulating the clavulanic acid fermentation yield of a strain.

[0006] In one embodiment, the above use is to provide a method for increasing the clavulanic acid fermentation yield of a strain, comprising the following steps: down-regulating, inactivating, weakening or knocking out the transcriptional regulatory factor gene SCLAV_p0126 (GeneBank No. EFG03617.1) in the genome of the clavulanic acid-producing bacteria.

[0007] Preferably, the clavulanic acid-producing bacterium is Streptomyces clavuligerus.

[0008] Furthermore, the clavulanic acid-producing bacterium is Streptomyces clavuligerus ATCC 27064, which may be referred to as a wild type (strain) herein.

[0009] The down-regulation, inactivation, functional attenuation or knockout of the above gene SCLAV_p0126 can be implemented in the following ways:

[0010] (1) Knockout the SCLAV_P0126 gene in the chromosome of the wild-type strain;

[0011] (2) down-regulating the expression level of gene SCLAV_P0126 in the chromosome of the wild-type strain;

[0012] (3) replacing the gene SCLAV_P0126 in the chromosome of the wild-type strain with a mutant of the gene SCLAV_P0126 that encodes a gene that has lost its function or is downregulated; and / or

[0013] (4) Block, inhibit or interfere with the expression of gene SCLAV_P0126 in the chromosome of the wild-type strain.

[0014] In one embodiment, the above method (2) is selected from the following group:

[0015] (2-1) mutation of the promoter region and / or coding region of the gene SCLAV_P0126 leads to downregulation of the expression level of the gene SCLAV_P0126;

[0016] (2-2) mutating the upstream regulatory factor of the gene SCLAV_P0126, resulting in downregulation of the expression level of the gene SCLAV_P0126; or

[0017] (2-3) Introduce the interacting protein of SCLAV_P0126 into the wild-type strain to change the function of the gene SCLAV_P0126.

[0018] Optionally, the mutation in the coding region in the above method (2-1) is a frameshift mutation, resulting in inactivation or loss of function of SCLAV_P0126.

[0019] The above methods (1), (2), (3) and / or (4) can be implemented through gene editing technology, antisense nucleic acid, and transcription regulation.

[0020] The above-mentioned gene editing technology can be selected from the following groups: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT.

[0021] The second aspect of the present invention provides an engineered bacterium of Streptomyces clavuligerus, in which the transcriptional regulatory factor gene SCLAV_p0126 (GeneBank No. EFG03617.1) in the genome is down-regulated, inactivated, functionally weakened or knocked out.

[0022] The third aspect of the present invention provides the use of the engineered Streptomyces clavuligerus in the production of clavulanic acid by fermentation.

[0023] The present invention first discovered that the transcriptional regulatory factor SCLAV_p0126 (GeneBank No. EFG03617.1) derived from Streptomyces clavuligerus has a negative regulatory effect on the synthesis of clavulanic acid by the strain. By knocking out the SCLAV_p0126 gene in Streptomyces clavuligerus through a genetic engineering method, a high-yield strain with a 113% increase in clavulanic acid production can be obtained, which has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the structural map of plasmid pB021-01 used to knock out the transcriptional regulatory factor SCLAV_p0126 in the genome of Streptomyces clavuligerus.

[0025] Figure 2 This is a schematic diagram of the construction of the SCLAV_p0126 gene knockout strain.

[0026] Figure 3 It is a bar graph comparing the clavulanic acid fermentation yield of the SCLAV_p0126 knockout mutant and the control strain. DETAILED DESCRIPTION

[0027] In our exploration of the genes or proteins related to the clavulanic acid metabolic pathway of Streptomyces clavuligerus, we tried to overexpress genes or proteins that positively regulate clavulanic acid biosynthesis and knock out genes or proteins that negatively regulate clavulanic acid biosynthesis. The results showed that after the SCLAV_p0126 gene of the wild-type Streptomyces clavuligerus ATCC 27064 was knocked out, the clavulanic acid fermentation yield of the mutant strain increased by more than double, indicating that downregulating the expression of specific genes is also an important strategy to increase the level of target metabolites.

[0028] In this article, for the sake of simplicity of description, a certain protein, such as the transcriptional regulatory factor SCLAV_p0126 (GeneBank No. EFG03617.1), is sometimes used interchangeably with the name of its encoding gene (DNA). Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for the transcriptional regulatory factor SCLAV_p0126, when used to describe the function or category of the transcriptional regulatory protein, it refers to the protein; when described as a gene, it refers to the gene encoding the protein.

[0029] The terms "Streptomyces clavuligerus (genetically) engineered bacteria", "engineered bacteria (strain)" and "mutant (bacteria) strain" herein have the same meaning and can be used interchangeably.

[0030] Correspondingly, for the convenience of description, the starting strain in which the gene SCLAV_p0126 has not been knocked out, such as Streptomyces clavuligerus ATCC 27064, can be referred to as the "wild type (strain)" or "starting strain".

[0031] As used herein, the terms "(clavulanic acid production) increase", "enhance" or "enhance" may mean an increase of at least 10% compared to a reference level (such as a starting strain / wild fungus), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including an increase of 100%, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0032] In the description of the technical solutions of the present invention, the terms used, such as "A and / or B", "A and / or B", "and / or", "and / or", are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0033] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0034] Example

[0035] The examples herein involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0036] In the examples herein, if no specific description is given for the reaction temperature or the operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0037] For experimental methods without specific conditions specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0038] Materials and methods

[0039] In the following examples, the plasmid pJTU1278 involved has been recorded in the SCI database document “Bu, XL; He, BB; Weng, JY; Jiang, CC; Zhao, YL; Li, SM; Xu, J.; Xu, MJ Constructing microbial hosts for the production of benzoheterocyclic derivatives. ACS Synth. Biol. 2020, 9 (9), 2282-2290”.

[0040] All primer synthesis and sequencing involved were completed by Shanghai Qingke Biotechnology Co., Ltd.

[0041] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0042] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0043] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium plus 20 g / L agar powder).

[0044] 2× spore pre-germination medium: yeast extract 10 g / L, casamino acid 10 g / L, calcium chloride 0.01 M, sterilized at 121°C for 20 min, used for spore germination culture in conjugation transfer experiments.

[0045] TES (0.05M, pH 8.0) solution: 1.15 g N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid was diluted to 100 mL pure water and the pH was adjusted to 8.0. Sterilized at 121°C for 20 min for later use. Used for conjugation transfer of Streptomyces clavuligerus spore germination.

[0046] Tryptone soy broth (TSB) culture: 3% TSB, for the culture of Streptomyces clavuligerus.

[0047] MS medium: 20g / L soybean cake powder infusion, 20g / L mannitol, 20g / L agar powder, used for conjugation transfer of rod-shaped Streptomyces culture.

[0048] Seed culture medium: 24g / L soybean superfine powder, 10g / L corn starch, 5g / L yeast autolysate, 0.8g / L potassium dihydrogen phosphate, adjust pH to 6.9-7.1. Dispense into 250mL shake flasks, 50mL of liquid per bottle, add 0.5mL of triglyceride to each bottle, sterilize at 121℃ for 20min, and use for shake flask seed culture.

[0049] Shake flask medium: 36g / L soybean superfine powder, 10g / L corn starch, 12g / L soybean protein extract, 1.95g / L potassium hydrogen phosphate, 1.5g / L potassium chloride, 1.0g / L magnesium chloride hexahydrate, 0.38g / L calcium chloride dihydrate, 0.18g / L sodium chloride, 0.08g / L ferric chloride hexahydrate, 0.02g / L copper chloride, 0.01g / L zinc chloride, 200mL / L MOPS solution, adjust the pH to 6.9-7.1. Dispense into 250mL shake flasks, each bottle has 50mL of liquid, and add 0.8mL of triglyceride to each bottle. Sterilize at 121℃ for 20min and use for shake flask fermentation culture. Among them, MOPS solution: 42g MOPS is added to 50mL of purified water, and then 88.5mL of 1M sodium hydroxide solution is added to 200mL, and the pH is adjusted to 7.1-7.3.

[0050] The gene SCLAV_p0126 knockout plasmid pB021-01 constructed in the example is preserved by Shanghai Banglin Biotechnology Co., Ltd. Any unit or individual can obtain the plasmid for verification of the present invention, but it cannot be used for other purposes, including development and utilization, scientific research and teaching, without the permission of Shanghai Banglin Biotechnology Co., Ltd.

[0051] Example 1: Knockout of the SCLAV_p0126 gene in Streptomyces clavuligerus ATCC 27064

[0052] This example is a process of constructing a mutant strain B021-01 that knocks out the expression of the SCLAV_p0126 gene. Figure 2 , specifically including the following steps:

[0053] Step 1: Construction of SCLAV_p0126 gene knockout plasmid pB021-01: Using the genomic DNA of Streptomyces clavuligerus ATCC 27064 as a template, primers up-F / up-R were used to amplify the upstream homology arm S1 of SCLAV_p0126, and primers down-F / down-R were used to amplify the downstream homology arm S2 of SCLAV_p0126. Through the one-step cloning method, it was cloned into the site of plasmid pJTU128 to obtain the knockout plasmid pB021-01. The plasmid map is shown in Figure 1 Its nucleotide sequence is shown in SEQ ID NO:3.

[0054] Plasmid pB021-01 can be used to knock out a 660 bp sequence in the coding region of gene SCLAV_p0126 of Streptomyces clavuligerus ATCC 27064, resulting in the gene being unable to encode normally and being inactivated.

[0055] The primer sequences used in the first step are:

[0056]

[0057] PCR system and conditions used for gene fragment preparation in the first step:

[0058] PCR reaction system: DNA template 30ng, primer 20pmol, 50% DMSO 4μL, PrimerSTAR max, add pure water to make up to 50μL;

[0059] PCR conditions: 95°C for 5 min; 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0060] Step 2: The knockout plasmid pB021-01 constructed in the first step was introduced into the recipient bacterium Streptomyces clavuligerus ATCC 27064 through conjugation transfer for site-specific recombination, and the correct conjugants were screened through resistance and PCR verification to obtain a mutant strain with SCLAV_p0126 gene knockout. Specifically, the following steps are included:

[0061] The gene knockout plasmid pB021-01 was transferred into Escherichia coli ET12567 (pUZ8002) to obtain the donor strain. The inoculated donor strain was cultured in LB medium containing final concentrations of 50 μg / mL ampicillin, 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, respectively, and cultured at 37°C with shaking for 20 hours. Subsequently, the bacteria were rinsed with fresh LB solution to remove residual antibiotics. Fresh spores of Streptomyces ATCC 27064 on the plate were collected, centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded and the spores were resuspended with 5 mL of TES solution (0.05 M, pH 8.0). The centrifuge tube was placed in a 50°C water bath for heat shock for 10 minutes, and then cooled to room temperature with tap water. 5 mL of 2× spore pre-germination medium was added and incubated at 37°C and 200 rpm for 3 hours. After incubation, the cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the spores were rinsed with fresh LB solution.

[0062] The treated spores were mixed with the donor strain at a volume ratio of about 1:10, evenly spread on MS solid medium containing 10mM MgCl2, and cultured at 37°C for 20 hours. Afterwards, thiostrepton (final concentration 12.5μg / mL) and nalidixic acid (final concentration 40μg / mL) were mixed in 1mL of sterile water, covered on the surface of MS solid medium, and transferred to a 30°C incubator for inverted culture after the medium dried. Usually after 3 to 5 days, conjugates will appear on the plate, which will be transferred to MS solid medium containing the same concentration of thiostrepton and nalidixic acid for further culture. The single-exchange strains were obtained by mycelial PCR verification screening. The obtained single-exchange strains were coated on MS plates without resistance and cultured at 30°C for 10 days. The spores on the plate were collected, diluted to a certain multiple, spread on non-resistant MS solid medium, and cultured at 30°C for 4 days to obtain a single clone. The single clone was identified by PCR using the identification primers checkF / checkR and sequenced to obtain a mutant strain with SCLAV_p0126 gene knockout and knockout plasmid eliminated, named B023-01.

[0063] In the second step, the PCR system and conditions used to screen mutants by PCR verification are as follows:

[0064] PCR system: DNA template 10-100 ng, primer 10 pmol, 50% DMSO 2 μL, 2×Mix buffer 10 μL, add pure water to make up to 20 μL;

[0065] PCR conditions: 95°C for 10 min; 9°C for 15 s, 60°C for 15 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0066] The primer sequences used in the second step are:

[0067]

[0068] Example 2: Strain fermentation

[0069] This example investigates the process of fermentation of clavulanic acid by mutant strain B021-01 with knockout SCLAV_p0126 gene, and compares it with wild-type strain ATCC 27064. The specific steps are as follows: mutant strain B021-01 with knockout SCLAV_p0126 gene and Streptomyces clavuligerus ATCC 27064 are respectively coated on solid MS medium for activation, cultured at 30°C for 14 days, scrape half of the spores and inoculate them into seed medium, cultured at 25°C and 200-250rpm for 48h, transferred to fermentation medium at an inoculum amount of 10% v / v, fermented at 25°C and 200-250rpm for 120h, and collected the fermentation broth for clavulanic acid detection.

[0070] Example 3: Detection of clavulanic acid content in fermentation broth

[0071] This embodiment is a method for detecting clavulanic acid using liquid phase. Specifically, 1 mL of fermentation broth is taken, centrifuged at 12000 rpm for 10 min, the supernatant is taken, appropriately diluted, filtered through a membrane, and then subjected to liquid phase detection.

[0072] Clavulanic acid detection conditions: Agilent 1260 liquid chromatograph, chromatographic column C18 (4.6×250 mm, 5 μm), mobile phase: mobile phase A (10 mmol tetrabutylammonium hydrogen sulfate, 11.5% acetonitrile, pH=6.0): mobile phase C (acetonitrile) = 90:10 (V / V), detection wavelength: 210 nm, column temperature: 33° C., flow rate: 1 ml / min, injection volume: 20 μl.

[0073] The comparison of clavulanic acid content in the fermentation broth of mutant strain B021-01 and Streptomyces clavuligerus ATCC 27064 is shown in Figure 3 The results showed that the clavulanic acid production of mutant strain B021-01 was increased by 113% compared with wild strain ATCC 27064, confirming that knocking out the SCLAV_p0126 gene can improve the clavulanic acid fermentation level of the strain.

[0074] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments and features in the embodiments herein can be combined with each other at will.

Claims

1. Use of the transcriptional regulatory factor SCLAV_p0126 (GeneBank No. EFG03617.1) in regulating the clavulanic acid fermentation yield of the strain.

2. The use according to claim 1, which is a method for increasing the clavulanic acid fermentation yield of a strain, characterized in that: The method comprises the following steps: down-regulating, inactivating, weakening or knocking out the expression of the transcriptional regulatory factor gene SCLAV_p0126 (GeneBank No. EFG03617.1) in the genome of clavulanic acid producing bacteria.

3. The use according to claim 2, characterized in that The clavulanic acid producing bacterium is Streptomyces clavuligerus.

4. The use according to claim 3, characterized in that The clavulanic acid producing bacterium is Streptomyces clavuligerus ATCC 27064.

5. The use according to claim 2, characterized in that The downregulation, inactivation, functional attenuation or knockout of the gene SCLAV_p0126 is carried out by: (1) Knockout the SCLAV_P0126 gene in the chromosome of the wild-type strain; (2) down-regulating the expression level of gene SCLAV_P0126 in the chromosome of the wild-type strain; (3) replacing the gene SCLAV_P0126 in the chromosome of the wild-type strain with a mutant of the gene SCLAV_P0126 that encodes a gene that has lost its function or is downregulated; and / or (4) Block, inhibit or interfere with the expression of gene SCLAV_P0126 in the chromosome of the wild-type strain.

6. The use according to claim 5, characterized in that Method (2) is selected from the following group: (2-1) mutation of the promoter region and / or coding region of the gene SCLAV_P0126 leads to downregulation of the expression level of the gene SCLAV_P0126; (2-2) mutating the upstream regulatory factor of the gene SCLAV_P0126, resulting in downregulation of the expression level of the gene SCLAV_P0126; or (2-3) Introduce the interacting protein of SCLAV_P0126 into the wild-type strain to change the function of the gene SCLAV_P0126.

7. The use according to claim 6, characterized in that The mutation in the coding region in embodiment (2-1) is a frameshift mutation, which results in inactivation or loss of function of SCLAV_P0126.

8. The use according to claim 5, characterized in that The methods (1), (2), (3) and / or (4) are implemented through gene editing technology, antisense nucleic acid, and transcription regulation.

9. An engineered strain of Streptomyces clavuligerus, characterized in that: The expression of the transcriptional regulatory factor gene SCLAV_p0126 (GeneBank No. EFG03617.1) in the genome is downregulated, inactivated, weakened or knocked out.

10. Use of the engineered strain of Streptomyces clavuligerus as claimed in claim 9 in the production of clavulanic acid by fermentation.

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