A method for increasing thaxtomin A production by using relA gene of Streptomyces scabii

Abstract

The present invention relates to the field of genetic engineering technology, and provides a method for increasing the yield of thaxtomin A by using the relA gene of Streptomyces scabii, firstly overexpressing the relA gene in Streptomyces scabii by a genetic engineering method to obtain a high-yield strain of thaxtomin A of Streptomyces scabii, and then using the obtained high-yield strain to ferment and produce thaxtomin A; wherein the nucleotide sequence of the relA gene is shown in SEQ ID NO.1. The present invention identifies the GTP pyrophosphate kinase gene relA that is positively correlated with the biosynthesis of thaxtomin A, and overexpressing the relA gene in Streptomyces scabii 87.22 by a genetic engineering approach to obtain a high-yield strain of thaxtomin A, thereby providing technical support for increasing the fermentation yield of thaxtomin A in industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for increasing the yield of thaxtomin A by using the relA gene of Streptomyces scabies. Background Art

[0002] Streptomyces is an important microbial resource that can produce secondary metabolites. It is closely related to humans, and its metabolites are widely used in medicine and health, animal husbandry, industry, and agricultural herbicides.

[0003] In Streptomyces, the synthesis of secondary metabolites is usually subject to the complex and rigorous regulation of one or more transcription factors. Transcriptional regulatory factors sense specific upstream signal molecules in the cell and then start specific regulatory actions, forming a multi-level regulatory network with pathway specificity, timing, and cross-cascades, which ultimately affects the synthesis of secondary metabolites. By modifying the biosynthetic pathway of specific signal molecules, the content of intracellular signal molecules can be increased, thereby achieving high yields of secondary metabolites.

[0004] Traditional high-yield industrial strains are obtained by random physical or chemical mutagenesis. Among them, random mutagenesis technology has uncertainty and cannot provide rational guidance for breeding. The purpose of the present invention is to obtain a thaxtomin A high-yield strain by directing gene modification through genetic engineering for the production of thaxtomin A.

[0005] Thaxtomin A has great potential for application in agricultural herbicides. Compared with commercial herbicides such as glyphosate, thaxtomin A has significantly better herbicidal activity against broadleaf weeds, and thaxtomin A has almost no damage to gramineous crops such as barley, rice and corn. Therefore, increasing the yield of thaxtomin A has gradually attracted widespread attention. Thaxtomin A can cause plant cell hypertrophy at the nanomolar level, and plant cells cannot elongate normally, resulting in stunted seedling development. Its main mechanism is to inhibit the production of cellulose. The biosynthetic gene cluster of Thaxtomin A is composed of seven genes: txtC, txtH, txtB, txtA, txtR, txtE and txtD. Among them, six genes (txtA, txtB, txtC, txtD, txtE, txtH) encode biosynthetic enzymes, and txtR encodes AraC / XylS family transcriptional regulatory proteins. txtR activates the expression of genes txtA, txtB, txtC and txtD, thereby activating the biosynthesis of thaxtomin A.

[0006] The intracellular derivative of GTP, tetraphosphate or pentaphosphate guanosine nucleoside (p)ppGpp, can produce a variety of effects on the regulation of cell life activities, such as sensing bacterial density, cell differentiation, bacterial toxin production, and pathogenicity of pathogens. Among them, (p)ppGpp is an "alarm" signal molecule for prokaryotes to respond to different environmental stresses and initiate the regulation of related physiological adaptive gene expression. When bacteria face amino acid starvation, unloaded tRNA appears at the protein synthesis A site in their ribosomes. This signal can be sensed by the RelA protein bound to the ribosome L11 protein. RelA can catalyze the pyrophosphorylation of GTP to synthesize the signal molecule (p)ppGpp.

[0007] So far, the mechanism of (p)ppGpp-mediated transcriptional regulation in model bacteria such as Escherichia coli has been widely studied, but there are very few studies on (p)ppGpp in actinomycetes that produce microbial drugs. Based on the biological information prediction of the genome sequence of Streptomyces scabii, the relA (SCAB_74961) gene of Streptomyces scabii encodes a GTP pyrophosphokinase RelA, which is responsible for catalyzing the pyrophosphorylation reaction of GTP to generate (p)ppGpp, and the laboratory research of the present invention found that the relA gene of Streptomyces scabii has a positive correlation with the biosynthesis of thaxtomin A. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for increasing the yield of thaxtomin A by using the relA gene of Streptomyces scabii.

[0009] The present invention adopts the following technical solutions to solve the above technical problems:

[0010] A method for increasing the yield of thaxtomin A by using the relA gene of Streptomyces scabii. The method comprises firstly overexpressing the relA gene in Streptomyces scabii by a genetic engineering method to obtain a high-yield strain of thaxtomin A of Streptomyces scabii, and then using the obtained high-yield strain to ferment and produce thaxtomin A; wherein the nucleotide sequence of the relA gene is shown in SEQ ID NO.1.

[0011] As one of the preferred embodiments of the present invention, in the step of overexpressing the relA gene in Streptomyces scabii by genetic engineering, the Streptomyces scabii strain 87.22 is used. The Streptomyces scabii strain 87.22 is a strain that has been disclosed and obtained by the public, provided by the China General Microbiological Culture Collection Center, and the collection number is CGMCC 4.1765.

[0012] As one of the preferred embodiments of the present invention, the relA gene is used to encode GTP pyrophosphokinase RelA; the amino acid sequence of the GTP pyrophosphokinase RelA is shown in SEQ ID NO.2, which is responsible for catalyzing the pyrophosphorylation reaction of GTP to generate (p)ppGpp.

[0013] As one of the preferred embodiments of the present invention, the expression product of the relA gene is used to positively regulate the biosynthesis of thaxtominA.

[0014] The advantages of the present invention compared to the prior art are:

[0015] In the present invention, the GTP pyrophosphokinase gene relA, which is positively correlated with the biosynthesis of thaxtomin A, was identified in the study. By overexpressing the relA gene in Streptomyces scabii 87.22 through genetic engineering, a high-yield strain of thaxtominA can be obtained, providing technical support for increasing the fermentation yield of thaxtomin A in industrial production. Among them, when the relA gene was knocked out in Streptomyces scabii 87.22, the thaxtomin A yield decreased significantly by 80.5%; and when the relA gene was complemented in the ΔrelA deletion mutant, the thaxtomin A yield was restored; thus indicating that the relA gene is positively correlated with the biosynthesis of thaxtomin A. When the copy number of the relA gene was increased in Streptomyces scabii 87.22, the thaxtomin A yield was significantly increased by 59.6% compared with the control strain, indicating that the overexpression of the relA gene in Streptomyces scabii can construct a high-yield strain of thaxtomin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the construction process of the relA gene deletion mutant strain ΔrelA (using the suicide plasmid pUCTSR and homologous recombination technology to delete 1644 bp within the relA gene on the chromosome of Streptomyces scabii);

[0017] Figure 2It is a double enzyme digestion verification diagram of the recombinant plasmids pUCTSR-ΔrelA-UP and pUCTSR-ΔrelA used for relA gene knockout (Figure A is the double enzyme digestion verification result of pUCTSR-ΔrelA-UP, in the figure, the "upper band" of lanes 1 and 2 represents the pUCTSR plasmid fragment of 4260bp, the "lower band" represents the upstream fragment of the relA gene of 1500bp, and M represents a 5000bp DNA Marker; Figure B is the double enzyme digestion verification result of pUCTSR-ΔrelA, in the figure, the "upper band" of lanes 1 and 2 represents the pUCTSR-ΔrelA-UP plasmid fragment of 5760bp, the "lower band" represents the downstream fragment of the relA gene of 1500bp, and M represents a 5000bp DNA Marker);

[0018] Figure 3 is the PCR verification graph of the mutant strain ΔrelA (in the graph, lane 1 represents the positive control 1475 bp; lane 2 represents the negative control 1759 bp; lane 3 represents the PCR amplification band of the positive clone, which is the same as 1; M represents a 5000 bp DNA Marker);

[0019] Figure 4 It is a double enzyme digestion verification diagram of the recombinant plasmid pIB-relA used for relA gene complementation and overexpression (in the figure, the "upper band" of lanes 1 and 2 represents the pIB139 plasmid fragment of 5900 bp, the "lower band" represents the relA gene fragment of 2544 bp, and M represents a 5000 bp DNA marker);

[0020] Figure 5 It is the PCR verification diagram of the complemented strain ΔrelA / pIB-relAΔrelA (in the figure, lane 1 represents the internal fragment of the apr resistance gene on the pIB139 plasmid, i.e., the positive control 750 bp; lanes 3 and 4 represent the PCR amplification bands of the screened positive clones, which are the same as 1; M represents a 5000 bp DNA marker);

[0021] Figure 6 It is an analysis chart of thaxtomin A production of a series of Streptomyces scabies strains (specifically including HPLC analysis of thaxtomin A production in fermentation broth of the starting strain 87.22, the mutant strain ΔrelA, the complemented strain ΔrelA / pIB-relA and the empty control strain ΔrelA / pIB139);

[0022] Figure 7 It is the transcriptional analysis diagram of thaxtomin A biosynthesis-related genes in the ΔrelA mutant (specifically including the regulatory gene txtR and the synthetic genes txtA and txtE in the thaxtomin A biosynthesis gene cluster);

[0023] Figure 8 It is the PCR verification diagram of the relA gene overexpression strain 87.22 / pIB-relA (in the figure, lane 1 represents the internal fragment of the apr resistance gene on the pIB139 plasmid, i.e., the positive control 750 bp; lanes 3 and 4 represent the PCR amplification bands of the screened positive clones, which are the same as 1; M represents a 5000 bp DNA marker);

[0024] Fig. 9 It is a graph of thaxtomin A production analysis of the overexpression strain 87.22 / pIB-relA (specifically including HPLC analysis of thaxtomin A production in the fermentation broth of the starting strain 87.22, the overexpression strain 87.22 / pIB-relA and the empty control strain 87.22 / pIB139). DETAILED DESCRIPTION

[0025] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0026] The strains and plasmids used in the following examples are shown in Table 1, and the primer sequences used are shown in Table 2.

[0027] The Escherichia coli used in the following examples were cultured in liquid LB medium or on LB solid plates supplemented with 2.0% agar at 37°C. The scab Streptomyces 87.22 strain was cultured in TSB medium or on SFM solid plates containing 2.0% agar at 28°C. The medium used for the fermentation of thaxtomin A by scab Streptomyces was OBB liquid fermentation medium, which was cultured at 28°C. The general operation techniques of Escherichia coli and scab Streptomyces were in accordance with standard operations. Primer synthesis and DNA sequencing were completed by General Biosystems (Anhui) Co., Ltd.

[0028] Table 1 Strains and plasmids used in the examples of the present invention

[0029]

[0030]

[0031] Table 2 Primers used in the examples of the present invention

[0032]

[0033] Example 1

[0034] relA gene related information:

[0035] For the location information of the relA (SCAB_74961) gene on the chromosome of Streptomyces scabii, please refer to NCBI. The nucleotide sequence of the relA gene is shown in SEQ ID NO.1, which is used to encode GTP pyrophosphokinase RelA; the amino acid sequence of the GTP pyrophosphokinase RelA is shown in SEQ ID NO.2, which is responsible for catalyzing the pyrophosphorylation reaction of GTP to generate (p)ppGpp.

[0036] Example 2

[0037] Construction of relA gene deletion mutant strain ΔrelA:

[0038] To construct the relA gene deletion mutant strain ΔrelA of Streptomyces scabies, see Figure 1 The internal sequence of the relA gene was replaced by the thiostrepton resistance gene tsr carried by the suicide plasmid pUCTSR.

[0039] Using the genome of Streptomyces scabies as a template, relA-UF / relA-UR and relA-DF / relA-DR were used as primers to PCR amplify 1500 bp of homology arm DNA fragments upstream and downstream of the relA gene. The specific primer sequences are shown in Table 2, where the underlined "AAGCTT", "TCTAGA", "GGATCC" and "GAATTC" sequences are the restriction sites of restriction endonucleases HindIII, XbaI, KpnI and EcoRI, respectively.

[0040] The upstream DNA fragment was recovered and treated with HindIII and XbaI for double restriction digestion, and then cloned into the pUCTSR plasmid to obtain the recombinant plasmid pUCTSR-ΔrelA-UP. The pUCTSR-ΔrelA-UP was double-digested for verification ( Figure 2 A); then, the downstream DNA fragment was recovered and treated with KpnI and EcoRI for double restriction digestion, and then cloned into the pUCTSR-ΔrelA-UP plasmid to obtain the recombinant plasmid pUCTSR-ΔrelA, and pUCTSR-ΔrelA was double-digested for verification ( Figure 2 B).

[0041] The verified recombinant plasmid pUCTSR-ΔrelA was transferred into Escherichia coli ET12567 (pUZ8002), and pUCTSR-ΔrelA was introduced into Streptomyces scabies 87.22 by cross-genus conjugation transfer technology. Thiostrepton was used to screen the resistant conjugants, and the selected strains were analyzed by PCR using primers relA-TF / relA-TR. The successfully verified positive strain was named ΔrelA ( Figure 3). Specific primer sequences are shown in Table 2.

[0042] Example 3

[0043] Construction of relA gene complemented strain ΔrelA / pIB-relA:

[0044] In order to complement the relA gene in the ΔrelA mutant, the genome of Streptomyces scabii was used as a template and relA-CF / relA-CR was used as primers to PCR amplify the complete DNA fragment of the relA gene. The specific primer sequences are shown in Table 2, where the underlined "CATATG" and "TCTAGA" sequences are the restriction endonuclease cutting sites of NdeI and XbaI, respectively.

[0045] The complete DNA fragment of the relA gene was connected to the plasmid pIB139 by restriction digestion to construct the recombinant plasmid pIB-relA. The pIB-relA was double-digested and verified ( Figure 4 ). The recombinant plasmid pIB-relA was transformed into ET12567 (pUZ8002), and then pIB-relA was introduced into the deletion mutant ΔrelA by conjugation transfer. Apramycin was used to screen the resistant conjugates, and apr-F / apr-R were used as primers for PCR verification. The correct one was the complemented strain ΔrelA / pIB-relA ( Figure 5 Using the same method, the empty vector pIB139 was introduced into the mutant strain ΔrelA to construct the empty vector control strain ΔrelA / pIB139 ( Figure 5 ). Specific primer sequences are shown in Table 2.

[0046] Example 4

[0047] Detection of thaxtomin A production in Streptomyces scabies strains:

[0048] The spores of Streptomyces scabii 87.22 and ΔrelA series strains growing in the same manner on the plate were inoculated into TSB seed bottles, shaken and cultured at 220 rpm and 28°C for 2 days, and then transferred to OBB liquid medium and continued to shake and culture at 220 rpm and 28°C for 7 days. After the fermentation was completed, thaxtomin A in the fermentation broth was extracted and analyzed by HPLC, and the yield ( Figure 6 ).

[0049] Example 5

[0050] Transcription analysis of related genes in ΔrelA:

[0051] The RNA of Streptomyces scabii 87.22 and mutant ΔrelA during fermentation was extracted using TaKaRa RNA extraction kit. After RNA was converted into cDNA, the transcription level of thaxtomin A biosynthesis-related genes was analyzed using real-time fluorescence quantitative PCR ( Figure 7 ).

[0052] Example 6

[0053] Overexpression of the relA gene in Streptomyces scabies 87.22:

[0054] The recombinant plasmid pIB-relA and the control plasmid pIB139 were introduced into Streptomyces scabii 87.22 by conjugation transfer technology, and the relA gene overexpression strain 87.22 / pIB-relA and the control strain 87.22 / pIB139 were obtained by the same apramycin screening and PCR verification method as mentioned above. Figure 8 ). The same method as above was used to ferment the 87.22 / pIB-relA overexpression strains, and the production of thaxtomin A in each strain was analyzed by HPLC ( Fig. 9 ).

[0055] Example 7

[0056] Analysis of specific experimental results of the above embodiments:

[0057] 1. The production of thaxtomin A decreased significantly after the deletion of relA gene

[0058] ΔrelA was fermented in OBB liquid medium for 7 days, and the thaxtomin A production was analyzed by HPLC after extraction. The production of ΔrelA was 80.5% lower than that of the original strain 87.22. Figure 6 ), indicating that the relA gene has a positive correlation with the biosynthesis yield of thaxtomin A.

[0059] 2. relA gene complementation

[0060] In order to confirm that the phenotype of the mutant strain ΔrelA is completely caused by the relA gene mutation, the present invention designed a relA gene complementation experiment for verification. pIB-relA uses the strong promoter PermE* of the erythromycin resistance gene to initiate the transcriptional expression of the relA gene, which is used to complement the mutant strain ΔrelA. After fermentation culture and HPLC detection, the thaxtomin A production of the complemented strain ΔrelA / pIB-relA was restored to a level comparable to that of the original strain 87.22 ( Figure 6 ).

[0061] 3. The transcription of thaxtomin A biosynthesis genes decreased significantly after the relA gene was deleted

[0062] qRT-PCR results confirmed that compared with the original strain 87.22, the expression levels of the regulatory gene txtR, structural genes txtA and txtE on the thaxtomin A biosynthetic gene cluster in the ΔrelA mutant decreased by 53.7%, 62.9% and 74.8% at 24h, and significantly decreased by 55.4%, 43.1% and 65.3% at 48h. Figure 7 ), indicating that the loss of relA could cause a significant decrease in the transcription level of genes related to thaxtomin A biosynthesis.

[0063] 4. Overexpression of the relA gene leads to a significant increase in thaxtomin A production

[0064] According to the experimental results that the relA gene deletion caused a significant decrease in the thaxtomin A production in Streptomyces scabii, it was confirmed that there was a positive correlation between the relA gene and the thaxtomin A production. Therefore, it can be inferred that overexpression of the relA gene in Streptomyces scabii is likely to increase the thaxtomin A production. In the present invention, the expression vector pIB-relA constructed above was introduced into the original strain 87.22 to increase the copy number of the relA gene, and the overexpression strain 87.22 / pIB-relA was constructed.

[0065] Fermentation culture and HPLC detection showed that the thaxtomin A production of the overexpression strain 87.22 / pIB-relA was significantly increased by 59.6% compared with the control strain 87.22 / pIB139 ( Fig. 9 ), which confirmed that overexpression of the relA gene in Streptomyces scabies through genetic engineering can indeed increase the production of thaxtomin A.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Streptomyces scab relat A method for genetically increasing thaxtomin A production, characterized in that: First, the gene was overexpressed in Streptomyces scabii 87.22 with the accession number CGMCC4.1765 by genetic engineering method. relat Gene, obtain a high-yield strain of thaxtomin A of Streptomyces scabii, and then use the obtained high-yield strain to ferment and produce thaxtomin A; wherein, relat The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The method according to claim 1, wherein the Streptomyces scabies relat A method for genetically increasing thaxtomin A production, characterized in that: Said relat The gene is used to encode GTP pyrophosphokinase RelA; the amino acid sequence of the GTP pyrophosphokinase RelA is shown in SEQ ID NO.2, and is responsible for catalyzing the pyrophosphorylation reaction of GTP to generate (p)ppGpp.

3. The method according to claim 1 or 2, wherein the Streptomyces scabies relat A method for genetically increasing thaxtomin A production, characterized in that: Said relat The expression product of the gene is used to positively regulate the biosynthesis of thaxtomin A.

Images