Method for improving yield of thaxtomin A through streptomyces scabies SCAB78491 gene

Abstract

The invention relates to the technical field of genetic engineering, and provides a method for improving the yield of thaxtomin A through a streptomyces scabies SCAB78491 gene. The method comprises the following steps: deleting the SCAB78491 gene of the streptomyces scabies through a genetic engineering way, constructing a thaxtomin A high-yield strain, and carrying out fermentation production; the sequence of the SCAB78491 gene is as shown in SEQ ID NO. 1, and the coded amino acid sequence of the SCAB78491 gene is as shown in SEQ ID NO. 2. The SCAB78491 gene in streptomyces scabies is determined as the thaxtomin A biosynthesis negative regulation gene for the first time through screening and identification, and the SCAB78491 gene in the streptomyces scabies is directionally deleted to obtain a thaxtomin A high-yield engineering strain, so that the yield of the thaxtomin A is effectively increased, and a technical support is provided for industrial fermentation optimization.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to a method using *Streptomyces scabies*. SCAB_78491 Methods to increase the production of thaxtomin A through gene therapy. Background Technology

[0002] Streptomyces, a group of Gram-positive filamentous bacteria with high G+C content, possess the ability to biosynthesize a variety of complex and functionally diverse secondary metabolites. These metabolites are widely used in medicine, animal husbandry, and as herbicides in industry and agriculture. In Streptomyces, the genes responsible for the synthesis of secondary metabolites are concentrated in gene clusters within the genome. The expression activity of these gene clusters is precisely regulated by various regulatory genes, primarily including pathway-specific regulation, pleiotropic regulation, and global regulation. Therefore, targeted modification of regulatory genes can effectively regulate the synthesis efficiency of Streptomyces secondary metabolites. A deeper understanding of the molecular mechanisms of secondary metabolic regulation is crucial for constructing high-performance engineered strains using genetic engineering techniques and applying them in practical production. This invention is based on this idea, aiming to obtain high-yield strains of thaxtomin A through targeted modification of regulatory genes via genetic engineering, thereby meeting the production requirements of thaxtomin A.

[0003] Thaxtomin A has shown great application potential in the field of agricultural herbicides. Compared with traditional chemical herbicides such as glyphosate, thaxtomin A exhibits more pronounced herbicidal activity against broadleaf weeds and is virtually harmless to gramineous crops such as barley, rice, and corn. Therefore, how to increase the yield of thaxtomin A has become a hot research topic. From the perspective of its mechanism of action, thaxtomin A can induce plant cell hypertrophy and inhibit normal cell elongation at nanomolar concentrations, leading to delayed seedling development. Its core target is the inhibition of plant cellulose biosynthesis. From the perspective of its synthetic mechanism, the biosynthesis of thaxtomin A depends on a gene cluster consisting of 7 genes, of which 6 are synthetic genes (…). txtA , txtB , txtC , txtD , txtE , txtH ) Responsible for encoding the corresponding biosynthetic enzyme, 1 regulatory gene txtR Encoding an AraC / XylS family transcriptional regulatory protein, this protein can activate... txtA , txtB , txtC and txtD Gene expression initiates the biosynthesis of thaxtomin A.

[0004] Lrp (leucine response regulatory protein) family proteins are a class of transcriptional regulatory factors widely distributed in prokaryotes, playing crucial roles in the body and participating in the regulation of many important physiological processes, including amino acid metabolism, core metabolism, energy metabolism, and substance transport. Currently, researchers have conducted relatively systematic studies on the regulatory function of Lrp proteins in antibiotic-producing bacteria. However, in pathogenic Streptomyces such as *Streptomyces scabies*, the regulatory function and related molecular mechanisms of Lrp proteins still require further in-depth exploration. Meanwhile, research on increasing thaxtomin A production in *Streptomyces scabies* through genetic engineering is relatively scarce; existing reports only involve methods involving the deletion of regulatory factor genes. cebR or SCAB_ Lrp2 Indirectly improve txtR The expression level of the gene is increased, thereby increasing the production of thaxtomin A.

[0005] Based on bioinformatics analysis and prediction of the genome sequence of Streptomyces scabies, the team of this invention discovered... SCAB_ 78491 The gene encodes an Lrp family protein, and subsequent laboratory studies have further confirmed that it is missing in *Streptomyces scabies*. SCAB_ 78491 After gene administration, the production of thaxtomin A significantly increased, thus clarifying... SCAB_78491 It is a negative regulator involved in the biosynthesis of thaxtomin A.

[0006] Therefore, there is an urgent need to establish a method to regulate Streptomyces scabies. SCAB_78491 An effective method to increase thaxtomin A production through genetics. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for treating scabies caused by Streptomyces cerevisiae. SCAB_78491 Methods to increase the production of thaxtomin A through gene therapy.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A type of Streptomyces scabies SCAB_78491 The method to increase thaxtomin A production through gene editing first involves deleting the gene from Streptomyces scabies. SCAB_78491 Genes were used to construct a high-yield strain of thaxtomin A, and then the obtained high-yield strain was used for fermentation to produce thaxtomin A; SCAB_78491 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the genetic engineering approach employs suicide plasmid pUCTSR and homologous recombination technology to achieve the synthesis of Streptomyces scabies from Streptomyces scabies. SCAB_78491 The absence of genes.

[0010] As one of the preferred embodiments of the present invention, the Streptomyces scabies strain is specifically Streptomyces scabies strain 87.22, which is a currently disclosed strain that is available to the public and is provided by the China General Microbiological Culture Collection Center with the preservation number CGMCC 4.1765.

[0011] As one of the preferred embodiments of the present invention, the process conditions for fermentation production of thaxtomin A are as follows: spores of the high-yield strain of thaxtomin A are inoculated into TSB seed culture medium and cultured with shaking at 28°C and 220 rpm for 2 days, and then transferred to OBB liquid fermentation culture medium and cultured with shaking at 28°C and 220 rpm for 7 days.

[0012] As one of the preferred embodiments of the present invention, the SCAB_78491 The gene encodes an Lrp family transcriptional regulatory protein that negatively regulates the biosynthesis of thaxtomin A; by deleting the aforementioned... SCAB_78491 The gene was removed from its negative regulatory effect on thaxtomin A biosynthesis, and the gene cluster containing thaxtomin A biosynthesis was upregulated. txtR , txtA , txtE Increase the transcriptional level of genes and enhance the biosynthetic yield of thaxtomin A.

[0013] The advantages of this invention compared to the prior art are: This invention, through screening and identification, is the first to clearly identify Streptomyces scabies. SCAB_78491 The gene is a negative regulator of thaxtomin A biosynthesis. Based on this discovery, this invention uses genetic engineering technology to directionally delete the gene in *Streptomyces scabies*. SCAB_78491 The gene was successfully constructed to obtain a high-yield thaxtomin A engineered strain, which effectively increased the yield of thaxtomin A in Streptomyces scabies, providing technical support for optimizing the fermentation yield of thaxtomin A in industrial production.

[0014] Specific research results show that Streptomyces scabies is missing in Streptomyces scabies. SCAB_78491 When the gene was tested, the yield of thaxtomin A was significantly increased by 57.92% compared to the original strain; while in Δ SCAB_78491 Replacement in mutant strains SCAB_78491 After gene sequencing, the production of thaxtomin A was restored; simultaneously, molecular mechanism studies confirmed that... SCAB_78491Gene deletion can relieve its negative regulatory effect on thaxtomin A biosynthesis and significantly upregulate the gene cluster involved in thaxtomin A biosynthesis. txtR , txtA , txtE The transcriptional level of the gene, in turn, promotes the synthesis of thaxtomin A; the above results fully confirm that... SCAB_78491 It is a negative regulator of thaxtomin A biosynthesis, and its targeted deletion can be an effective means to increase the biosynthetic yield of thaxtomin A in Streptomyces scabii. Attached Figure Description

[0015] Figure 1 yes SCAB_78491 A diagram showing the location of the gene and its neighboring genes on the chromosome of Streptomyces scabies. Figure 2 This is a schematic diagram of constructing a deletion mutant using the suicide plasmid pUCTSR and homologous recombination technology; Figure 3 It is Δ SCAB_78491 Schematic diagram of PCR verification of deletion mutant strain (in the figure, the thiostreptin resistance gene is used). tsr Replacement of the chromosome of Streptomyces scabies SCAB_78491 (In this gene, M represents a 5000bp DNA Marker, 1 represents a positive control of 1660bp, 2 represents a negative control of 492bp, and 3 represents a positive clone whose PCR clone amplification band is the same size as 1). Figure 4 It is the original strain of Streptomyces scabies, strain 87.22, and Δ. SCAB_78491 Graph showing the yield analysis of thaxtomin A in fermentation products of deletion mutants and related strains (specifically including the original strain *Streptomyces scabica* 87.22, the deletion mutant Δ...). SCAB_78491 , replenishment strain Δ SCAB_78491 / pIB- 78491 and its empty vector control strain Δ SCAB_78491 HPLC analysis of thaxtomin A yield in fermentation broth of / pIB139; in the figure, "**" indicates p<0.01, and "***" indicates p<0.001). Figure 5 It is the original strain *Streptomyces scabica* 87.22 and the deletion mutant strain Δ SCAB_78491 Figure showing the results of cell dry weight analysis during fermentation; Figure 6 It is the original strain *Streptomyces scabica* 87.22, and the deletion mutant strain Δ. SCAB_78491 , replenishment strain Δ SCAB_ 78491 / pIB- 78491 and Δ SCAB_78491The spore growth of / pIB139 is shown in the figure (Figure A shows the sporulation at 24h of culture, Figure B shows the sporulation at 48h of culture, Figure C shows the sporulation at 72h of culture, and Figure D shows the location of the strain). Figure 7 It is the original strain *Streptomyces scabica* 87.22 and Δ SCAB_78491 Transcriptional analysis results of thaxtomin A biosynthesis-related genes in deletion mutants (Figure, A is shown in the figure). txtR Comparison of relative transcription levels of the gene at 24h and 48h, Figure B shows... txtA The relative transcriptional levels of the gene at 24h and 48h are compared in Figure C. txtE Comparison of relative transcription levels of the gene at 24h and 48h; where "**" indicates p<0.01 and "***" indicates p<0.001. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0017] The strains and plasmids used in the following examples are shown in Table 1, and the primer sequences used are shown in Table 2.

[0018] The *E. 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. *Streptomyces scabica* 87.22 was cultured in TSB medium or on SFM solid plates containing 2% agar at 28°C. The medium used for fermentation of *Streptomyces scabica* to produce thaxtomin A was OBB liquid fermentation medium, cultured at 28°C. General operating techniques for *E. coli* and *Streptomyces scabica* were performed according to standard operating procedures. Primer synthesis and DNA sequencing were performed by General Biosystems (Anhui) Co., Ltd.

[0019] TSB seed culture medium: 15g tryptone, 5g soybean peptone, 5g NaCl, distilled water to a final volume of 1000mL, pH adjusted to 7.2.

[0020] OBB liquid fermentation medium (oat bran medium): 30g oat bran, 1000mL tap water, boil and then simmer for 10 minutes, stirring constantly. Filter out impurities with gauze, bring the filtrate to a final volume of 1000mL, and dispense into 50mL containers.

[0021] Table 1. Strains and plasmids used in the embodiments of the present invention.

[0022] Table 2 Primers used in the embodiments of the present invention

[0023] Example 1 SCAB_78491 Gene-related information: SCAB_78491 For information on the location of the gene and its neighboring genes on the chromosome of *Streptomyces scabica*, see [link to relevant documentation]. Figure 1 .

[0024] SCAB_78491 The gene nucleotide sequence is shown in SEQ ID NO.1, and its encoded product is an Lrp family transcriptional regulatory protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0025] Example 2, Δ ​ Construction of deletion mutants (see) ​ ): Using the genome of Streptomyces scabies as a template, 78491 -UF / R and 78491 -DF / R was used as a primer for PCR amplification. ​ The upstream and downstream 1500bp homologous fragments of the gene were extracted, and the target fragment was recovered and used separately. ​ dIII / ​ I and ​ I / ​ The upstream and downstream fragments were digested with RI double enzymes, and the target fragment was recovered by agarose gel electrophoresis and cloned into the pUCTSR plasmid to obtain the recombinant plasmid pUCTSR-Δ. ​ For pUCTSR-Δ ​ Double enzyme digestion was performed for verification. The specific primer sequences are shown in Table 2, where the underlined sequences “AAGCTT”, “TCTAGA”, “GGATCC”, and “GAATTC” represent the restriction endonucleases, respectively. ​ dIII、 ​ I ​ and ​ The restriction enzyme site of RI.

[0026] The constructed recombinant plasmid was transformed into E. coli ET12567 (pUZ8002), and the plasmid pUCTSR-Δ was transferred using cross-generic conjugation transfer technology. ​ Transformed into *Streptomyces scabica* 87.22, resistant positive mutants were screened using thiotetracycline, and primers were used to... 78491 -CF / R performs PCR analysis on the selected strains, and the successfully verified positive strains are named Δ. ​ ( ​ ).

[0027] Example 3: Construction of the complement strain and the complement empty vector strain: Using the genome of Streptomyces scabies as a template, primers were used... 78491 -CF / R, PCR amplification of complete ​ 78491 Gene fragments, simultaneously introduced on both sides ​ RI and ​ dIII restriction site. The PCR product was recovered by agarose gel electrophoresis and ligated into the vector plasmid pIB139 to obtain the recombinant plasmid pIB- 78491 Double enzyme digestion confirmed successful construction of the recombinant plasmid. The recombinant plasmid pIB- 78491 The empty vector pIB139 was transformed into competent ET12567 (pUZ8002) cells, and after conjugation transfer, pIB- 78491 and pIB139 introduced deletion mutant Δ ​ In the process, resistant conjugates were screened using apramycin, and the correct complement strain Δ was obtained after PCR verification. ​ / pIB- 78491 and replenishment of empty vector strain Δ ​ 78491 / pIB139.

[0028] Example 4: Detection of thaxtomin A production in a series of Streptomyces strains causing scabies: The same growth patterns of Streptomyces scabii 87.22 and Δ on the plate were compared. ​ Spores of the series of strains were inoculated into TSB seed bottles and cultured on a shaker at 28°C and 220 rpm for 2 days. They were then transferred to OBB liquid fermentation medium and cultured at 28°C and 220 rpm for 7 days. After fermentation, thaxtomin A in the fermentation broth was extracted and analyzed by HPLC. The yield was calculated using a thaxtomin A standard curve.

[0029] Example 5, Streptomyces 87.22 and Δ ​ Transcriptional analysis of genes related to thaxtomin A biosynthesis: Using an RNA extraction kit, Streptomyces scabioides 87.22 and the mutant strain Δ were extracted during the fermentation process. ​ 78491 RNA was extracted and reverse transcribed into cDNA. The transcriptional level of genes related to thaxtomin A biosynthesis was analyzed using real-time quantitative PCR.

[0030] Example 6: Analysis of specific experimental results for each of the above examples: 1. ​ Gene deletion significantly increased thaxtomin A production. Δ ​ Fermentation was carried out in OBB liquid fermentation medium for 7 days. After extraction, HPLC analysis showed that the yield of thaxtomin A was 57.92% higher than that of the original strain 87.22. ​ ),show ​ The gene exhibits a negative regulatory relationship with thaxtomin A biosynthesis. During fermentation, samples of the fermenting cells were taken daily and biomass was measured. The results showed that Δ ​ The cell count of the deletion mutant was almost identical to that of the original strain 87.22. ​ ),show ​ The absence of this component has almost no effect on bacterial growth, i.e. ​ The absence of this component affects thaxtomin A production not by influencing cell growth.

[0031] 2. ​ Gene replacement To determine the deletion mutant Δ ​ The phenotype is entirely determined by ​ Caused by gene mutation, this invention constructs ​ Gene complementation experiment; pIB- 78491 Initiation using the strong promoter PermE* of the erythromycin resistance gene ​ Transcriptional expression of genes, used for mutant strain Δ ​ The replenishment was carried out. Fermentation and HPLC analysis revealed that the replenished strain Δ... ​ / pIB- 78491 The yield of thaxtomin A in the middle strain recovered to a level consistent with that of the original strain 87.22.

[0032] 3. ​ Gene deletion does not affect the morphological differentiation of the strain. To clarify ​ Whether genes affect bacterial spore formation was investigated by comparing the original strain 87.22 and the deletion mutant strain Δ. ​ , replenishment strain Δ ​ / pIB- 78491 and replenishment of empty vector strain Δ ​ / pIB139 was simultaneously spread on SFM plates and incubated upside down at 28°C to observe spore growth. The original strain 87.22 and the replenished strain Δ were compared. ​ / pIB- 78491 Spore morphology, Δ ​ and Δ ​ / pIB139 produces spores faster ( ​ The above results indicate that... ​ The absence of [something] promotes the formation of Streptomyces scabies spores.

[0033] 4. ​ Negative regulation of the transcription of the thaxtomin A biosynthesis gene The results of qRT-PCR showed that, compared with the starting strain 87.22, the deletion mutant Δ ​ In the middle, at 24h and 48h, the genes on the thaxtomin A biosynthesis gene cluster ​ , ​ , ​ The expression levels of all of them were significantly increased ( ​ ),show ​ The deletion of genes can lead to a significant increase in the transcriptional level of genes related to thaxtomin A biosynthesis, thereby increasing the production of thaxtomin A.

[0034] In summary, this invention clarifies the presence of *Streptomyces* species responsible for scabies. ​ This gene is a negative regulator of thaxtomin A biosynthesis, and a method to increase thaxtomin A production by deleting this gene is provided. Molecular mechanism studies show that this gene deletion can upregulate the thaxtomin A biosynthesis gene cluster. ​ , ​ , ​ The gene is transcribed at a low level without affecting cell growth, and yield can be restored to the original level after gene restoration. Furthermore, gene deletion can promote spore formation in the strain, facilitating large-scale fermentation. This invention is simple to operate and allows for precise control, providing key technical support for the industrial-scale, efficient production of thaxtomin A, and has significant agricultural application value.

[0035] 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 within the protection scope of the present invention.

Claims

1. A method of treatment using *Streptomyces scabies* SCAB_78491 A method for increasing thaxtomin A production using genes, characterized by: First, the gene in Streptomyces scabies was deleted through genetic engineering. SCAB_78491 Genes were used to construct a high-yield strain of thaxtomin A, and then the obtained high-yield strain was used for fermentation to produce thaxtomin A; SCAB_78491 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence it encodes is shown in SEQ ID NO.

2.

2. The method according to claim 1, using *Streptomyces scabies* SCAB_78491 A method for increasing thaxtomin A production using genes, characterized by: The genetic engineering approach utilizes suicide plasmid pUCTSR and homologous recombination technology to achieve the desired effect in Streptomyces scabies. SCAB_78491 The absence of genes.

3. The method according to claim 1, using *Streptomyces scabies* SCAB_78491 A method for increasing thaxtomin A production using genes, characterized by: The specific Streptomyces scabies strain mentioned is Streptomyces scabies strain 87.

22.

4. The method according to claim 1, using *Streptomyces scabies* SCAB_78491 A method for increasing thaxtomin A production using genes, characterized by: The process conditions for fermenting thaxtomin A are as follows: spores of the high-yield thaxtomin A strain are inoculated into TSB seed medium and cultured at 28°C and 220 rpm for 2 days with shaking, and then transferred to OBB liquid fermentation medium and cultured at 28°C and 220 rpm for 7 days with shaking.

5. The method according to any one of claims 1 to 4, via Streptomyces scabies SCAB_78491 A method for increasing thaxtomin A production using genes, characterized by: The SCAB_78491 The gene encodes an Lrp family transcriptional regulatory protein that negatively regulates the biosynthesis of thaxtomin A; by deleting the aforementioned... SCAB_78491 The gene was removed from its negative regulatory effect on thaxtomin A biosynthesis, and the gene cluster containing thaxtomin A biosynthesis was upregulated. txtR , txtA , txtE Increase the transcriptional level of genes and enhance the biosynthetic yield of thaxtomin A.

Images