Method for improving yield of thaxtomin A through streptomyces scabies SCAB82261 gene

Abstract

The invention provides a method for improving the yield of thaxtomin A through a streptomyces scabies SCAB82261 gene. The method comprises the following steps: deleting the streptomyces scabies SCAB82261 gene through genetic engineering to obtain a thaxtomin A high-yield strain, and carrying out fermentation production; wherein the nucleotide sequence of the SCAB82261 gene is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2. The invention has the following advantages: the thaxtomin A biosynthesis negative regulation gene SCAB82261 is screened, and the PaaK protein encoded by the thaxtomin A biosynthesis negative regulation gene SCAB82261 can catalyze the degradation of aromatic compounds such as phenylalanine; when the streptomyces scabies SCAB82261 gene is deleted, a high-yield strain of the thaxtomin A can be obtained, and a technical support is provided for improving the yield of industrial production of the thaxtomin A.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to a method using *Streptomyces scabies*. SCAB_82261 Methods to increase the production of thaxtomin A through gene therapy. Background Technology

[0002] Streptomyces are a group of Gram-positive filamentous bacteria with high G+C content. Their core characteristic lies in their ability to biosynthesize a large number of structurally complex and functionally diverse secondary metabolites. These products have been widely used in many important fields such as medicine and health, animal husbandry, industrial production, and agricultural herbicides, making them a key research object for the development and utilization of microbial resources. In Streptomyces, the genes for the synthesis of secondary metabolites are usually distributed in tandem within the genome in the form of gene clusters. The expression activity of these gene clusters is regulated by multiple regulatory mechanisms, mainly including pathway-specific regulation, pleiotropic regulation, and global regulation. Optimizing metabolism through targeted modification of key regulatory genes has become a core technical approach for obtaining engineered strains that produce high yields of target products. In-depth analysis of the molecular mechanisms of secondary metabolic regulation is of great significance for promoting the application of genetic engineering technology in the industrial production of microbial products. Based on this, this invention aims to construct a high-yield thaxtomin A strain by targeted modification of key genes through genetic engineering, providing technical support for its large-scale production.

[0003] Thaxtomin A, an important secondary metabolite derived from Streptomyces, has shown significant application potential in the field of agricultural herbicides. Compared with traditional commercial herbicides such as glyphosate, thaxtomin A exhibits superior herbicidal activity against broadleaf weeds and is virtually harmless to gramineous crops such as barley, rice, and corn, aligning with the core needs of modern agriculture for "precision weed control and crop protection." Therefore, increasing the yield of thaxtomin A has become a research hotspot in this field. From a mechanism of action perspective, thaxtomin A can induce abnormal hypertrophy of plant cells and inhibit normal cell elongation at nanomolar concentrations, ultimately leading to delayed seedling development in weeds. Its molecular mechanism primarily achieves its weeding effect by blocking the biosynthesis of plant cellulose. From a biosynthetic perspective, the synthesis of thaxtomin A depends on... txtC , txtH , txtB , txtA , txtR , txtE and txtD Gene clusters composed of genes, among which txtA , txtB , txtC , txtD , txtE , txtH Encoding biosynthetic enzymes, txtREncoding AraC / XylS family transcriptional regulatory proteins, txtR activating genes txtA , txtB , txtC and txtD expression, thus activating the biosynthesis of thaxtomin A. In addition, tryptophan, arginine, and phenylalanine are essential amino acid precursors for the synthesis of thaxtomin A. In theory, increasing the intracellular accumulation of such precursor amino acids can directly promote the biosynthesis efficiency of thaxtomin A, which provides a clear direction for improving the yield of target products through metabolic regulation.

[0004] The phenylacetic acid degradation pathway is the core pathway for the metabolism of aromatic compounds in organisms. The upstream metabolic pathways of different types of aromatic compounds will ultimately converge on the common intermediate phenylacetyl-CoA, which enters the tricarboxylic acid (TCA) cycle for degradation after being metabolically converted into succinyl-CoA and acetyl-CoA. In this process, the conversion of aromatic compounds to phenylacetyl-CoA first depends on the catalysis of phenylacetic acid-CoA ligase (PaaK), and the subsequent steps are completed through the oxidative metabolism of the phenylacetic acid degradation proteins PaaABCDE. However, currently, in actinomycetes producing microbial drugs, there are few studies on the function of the PaaK protein, and its regulatory role in the synthesis of secondary metabolites has not been clarified, which greatly restricts the technological development of optimizing the synthesis of target products through directional metabolic regulation.

[0005] Based on the genomic biology information prediction of Streptomyces scabiei, its SCAB_82261 gene encodes the PaaK protein, and it is speculated that this gene may affect the supply of intracellular precursor amino acids by catalyzing the degradation of aromatic compounds such as phenylalanine. Further functional verification experiments in our laboratory found that after the deletion of the SCAB_82261 gene in Streptomyces scabiei, the yield of thaxtomin A increased significantly. This result not only confirmed the functional speculation of the SCAB_82261 gene, but also revealed the regulatory mechanism of increasing the supply of precursors by blocking the phenylalanine degradation metabolism to improve the yield of thaxtomin A. Summary of the Invention

[0006] 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 SCAB_82261 gene of Streptomyces scabiei.

[0007] The present invention adopts the following technical solutions to solve the above technical problems: A method for increasing the yield of thaxtomin A by using the SCAB_82261 gene of Streptomyces scabiei, first through genetic engineering approaches in Streptomyces scabiei for SCAB_82261Gene deletion was performed to obtain a high-yielding strain of *Streptomyces thaxtominA* that produces scabies. The obtained strain was then used for fermentation to produce thaxtomin A. Wherein... SCAB_82261 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0008] As one of the preferred embodiments of the present invention, the method of treatment with Streptomyces scabies is described. SCAB_82261 In the gene deletion step, the specific *Streptomyces scabica* strain used was *Streptomyces scabica* strain 87.22. Gene deletion was achieved using the suicide plasmid pUCTSR and homologous recombination technology. *Streptomyces scabica* strain 87.22 is a publicly available strain, provided by the China General Microbiological Culture Collection Center (CGMCC) with the depositary number CGMCC 4.1765.

[0009] As one of the preferred embodiments of the present invention, in the step of fermenting to produce thaxtomin A, the fermentation process conditions 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.

[0010] As one of the preferred embodiments of the present invention, the SCAB_82261 The gene encodes the phenylacetic acid-CoA ligase PaaK, which catalyzes the degradation of aromatic compounds, including phenylalanine; by deleting the [specific gene]... SCAB_82261 The gene blocks the degradation and metabolism of phenylalanine, increasing the accumulation of phenylalanine in the cell, and simultaneously upregulates the thaxtomin A biosynthesis gene cluster. txtR , txtA , txtE The transcriptional level of the gene increases the biosynthetic yield of thaxtomin A (used as a herbicide).

[0011] The advantages of this invention compared to the prior art are: This invention has screened genes that negatively regulate thaxtomin A biosynthesis. SCAB_82261 This gene encodes phenylacetic acid-CoA ligase (PaaK). The PaaK protein can influence the supply of intracellular precursor amino acids by catalyzing the degradation of aromatic compounds such as phenylalanine. The specific reaction process by which it mediates the degradation of phenylalanine into the TCA cycle is as follows: Figure 1 As shown. This invention utilizes genetic engineering to target Streptomyces scabies. SCAB_82261 Gene deletion was performed to obtain a high-yield strain of thaxtomin A, providing technical support for improving the fermentation yield of thaxtomin A in industrial production.

[0012] Among them, when Streptomyces scabies is missing SCAB_82261 When the gene was activated, thaxtomin A production increased significantly by 110.5%; while in Δ SCAB_82261 Replacement in mutant strains SCAB_82261 Following gene sequencing, thaxtomin A production recovered; this indicates that SCAB_82261 is a negative regulator involved in thaxtomin A biosynthesis. SCAB_82261 Gene deletion can be used to target and increase the biosynthetic yield of thaxtomin A in Streptomyces scabies. Attached Figure Description

[0013] Figure 1 yes SCAB_82261 A diagram illustrating the degradation of phenylalanine into the TCA cycle by a gene-encoded protein. Figure 2 yes SCAB_82261 A diagram showing the location of the gene and its neighboring genes on the chromosome of Streptomyces scabies. Figure 3 This is a schematic diagram of constructing a deletion mutant using the suicide plasmid pUCTSR and homologous recombination technology; Figure 4 It is Δ SCAB_82261 Schematic diagram of PCR verification of deletion mutant strain (in the figure, the thiotetracycline resistance gene tsr is replaced on the chromosome of Streptomyces scabii). SCAB_82261 Gene, M represents a 5000bp DNA Marker, 1 represents a positive control of 1662bp, 2 represents a negative control of 1332bp, and 3 and 4 represent PCR clone amplification bands of positive clones that are the same size as 1). Figure 5 It is the original strain of Streptomyces scabies, strain 87.22, and Δ. SCAB_82261 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_82261 , replenishment strain Δ SCAB_82261 / pIB- 82261 and its empty vector control strain Δ SCAB_82261 HPLC analysis of thaxtomin A yield in fermentation broth of / pIB139; and in the figure, "*" indicates P<0.05, and "***" indicates p<0.001). Figure 6 It is the original strain of Streptomyces scabies, strain 87.22, and Δ. SCAB_82261 Biomass analysis diagram of the deletion mutant (specifically, cell dry weight analysis during fermentation). Figure 7It is the original strain of Streptomyces scabies, strain 87.22, and Δ. SCAB_82261 Analysis of spore growth of deletion mutants and related strains (specifically including the original strain *Streptomyces scabica* 87.22, and the deletion mutant Δ...). SCAB_82261 , replenishment strain Δ SCAB_82261 / pIB- 82261 and Δ SCAB_82261 Sporulation of / pIB139 on SFM plates). Figure 8 It is the original strain *Streptomyces scabica* 87.22 and Δ SCAB_82261 Transcriptional analysis of thaxtomin A biosynthesis-related genes in deletion mutants (specifically including the thaxtomin A biosynthesis gene cluster). txtR , txtA , txtE Transcriptional analysis at 24h and 48h; and in the figure, "**" indicates P<0.01, and "***" indicates p<0.001). Detailed Implementation

[0014] 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.

[0015] The strains and plasmids used in the following examples are shown in Table 1, and the primer sequences used are shown in Table 2.

[0016] 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.

[0017] TSB seed culture medium: 15g tryptone, 5g soybean peptone, 5g NaCl, distilled water to a final volume of 1000mL, pH adjusted to 7.2.

[0018] 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.

[0019] Table 1. Strains and plasmids used in the embodiments of the present invention.

[0020] Table 2 Primers used in the embodiments of the present invention

[0021] Example 1 SCAB_82261 Gene-related information: For information on the location of the gene and its neighboring genes on the chromosome of *Streptomyces scabica*, see [link to relevant documentation]. SCAB_82261 .

[0022] Figure 2 The gene nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0023] Example 2, Δ SCAB_82261 Construction of deletion mutants (see SCAB_82261 ): Using the genome of Streptomyces scabies as a template, 82261 -UF / R and 82261 -DF / R was used as a primer for PCR amplification. Figure 3 The upstream and downstream 1500bp homologous fragments of the gene were extracted, and the target fragment was recovered and used separately. SCAB_82261 dIII / Hin I and Xba I / Sam 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. Eco For pUCTSR-Δ SCAB_82261 Double enzyme digestion was performed for verification. The specific primer sequences are shown in Table 2, where the underlined sequences “AAGCTT”, “TCTAGA”, “CCCGGG”, and “GAATTC” represent the restriction endonucleases, respectively. SCAB_82261 dIII、 Hin I Xba and 、SamI The restriction enzyme site of RI.

[0024] The constructed recombinant plasmid was transformed into E. coli ET12567 (pUZ8002), and the plasmid pUCTSR-Δ was transferred using cross-generic conjugation transfer technology. Eco Transformed into *Streptomyces scabica* 87.22, resistant positive mutants were screened using thiotetracycline, and primers were used to... 82261 -CF / R performs PCR analysis on the selected strains, and the successfully verified positive strains are named Δ.SCAB_82261 ( SCAB_82261 ).

[0025] 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... 82261 -CF / R, PCR amplification of complete Figure 4 82261 Gene fragments, simultaneously introduced on both sides SCAB_ RI and Eco RV restriction site. The PCR product was recovered by agarose gel electrophoresis and ligated into the vector plasmid pIB139 to obtain the recombinant plasmid pIB- 82261 Double enzyme digestion confirmed the successful construction of the recombinant plasmid.

[0026] Recombinant plasmid pIB- 82261 The empty vector pIB139 was transformed into competent ET12567 (pUZ8002) cells, and after conjugation transfer, pIB- 82261 and pIB139 introduced deletion mutant Δ Eco In the middle stage, resistant conjugates were screened using apramycin, and the correct complement strain Δ was obtained after PCR verification. SCAB_82261 / pIB-82261 and the reintroduced empty vector strain Δ SCAB_82261 / pIB139.

[0027] 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. SCAB_82261 Series of strains (deletion mutant Δ) SCAB_82261 , replenishment strain Δ SCAB_82261 / pIB- 82261 and its empty vector control strain Δ SCAB_82261 Spores of (pIB139) were inoculated into TSB seed bottles and cultured on a shaker at 28°C and 220 rpm for 2 days. Then, they were transferred to OBB liquid 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.

[0028] Example 5, Streptomyces 87.22 and Δ SCAB_82261 Transcriptional analysis of genes related to thaxtomin A biosynthesis: Using the TaKaRa RNA extraction kit, Streptomyces scabii 87.22 and the mutant strain Δ were extracted during the fermentation process. SCAB_82261RNA was extracted and reverse transcribed into cDNA. The transcriptional level of genes related to thaxtomin A biosynthesis was analyzed using real-time quantitative PCR.

[0029] Example 6: Analysis of specific experimental results for each of the above examples: 1. SCAB_82261 Gene deletion significantly increased thaxtomin A production. Δ SCAB_82261 Fermentation in OBB liquid medium for 7 days, followed by extraction and HPLC analysis, showed that the yield of thaxtomin A was 110.5% higher than that of the original strain 87.22. SCAB_82261 ),show Figure 5 The gene showed a negative correlation with thaxtomin A biosynthesis. During fermentation, samples of the fermenting cells were taken daily and biomass was measured. The results indicated that Δ... SCAB_82261 82261 The cell count of the deletion mutant was almost identical to that of the original strain 87.22. SCAB_ ),show Figure 6 The absence of this component has almost no effect on bacterial growth, i.e. SCAB_82261 The absence of this component affects thaxtomin A production not by influencing cell growth.

[0030] 2. SCAB_82261 Gene replacement To determine the deletion mutant Δ SCAB_82261 The phenotype is entirely determined by SCAB_82261 Caused by gene mutation, this invention constructs SCAB_82261 Gene complementation experiment; pIB- 82261 Initiation using the strong promoter PermE* of the erythromycin resistance gene SCAB_82261 Transcriptional expression of genes, used in mutant strain Δ SCAB_82261 The replenishment was carried out. Fermentation and HPLC analysis revealed that the replenished strain Δ... SCAB_82261 / pIB- 82261 The yield of thaxtomin A recovered to a level consistent with that of the original strain 87.22. [[ID=ll1]]SCAB_82261 ).

[0031] 3. Figure 5 Gene deletion does not affect the morphological differentiation of the strain. To clarify SCAB_82261 Whether genes affect bacterial spore formation was investigated by comparing the original strain 87.22 and the deletion mutant strain Δ. SCAB_82261 , replenishment strain Δ SCAB_82261 / pIB- 82261 and replenishment of empty vector strain Δ SCAB_82261 / pIB139 was simultaneously spread on SFM plates and incubated upside down at 28°C to observe spore growth. The results showed that, compared to the original strain 87.22, the deletion mutant Δ SCAB_82261 , replenishment strain Δ SCAB_82261 / pIB- 82261 and replenishment of empty vector strain Δ SCAB_82261 Δ was found in the spore morphology of / pIB139. SCAB_82261 and Δ SCAB_82261 / pIB139 produces spores faster ( SCAB_82261 ),illustrate Figure 7 The absence of [something] promotes the formation of Streptomyces scabies spores.

[0032] 4. SCAB_82261 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 SCAB_82261 In the middle, at 24h and 48h, the genes on the thaxtomin A biosynthesis gene cluster SCAB_82261 , txtR , txtA The expression levels of all of them were significantly increased ( txtE ),show Figure 8 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.

[0033] In summary, this invention clearly states... SCAB_82261 The gene acts as a negative regulator of thaxtomin A synthesis, and its encoded PaaK protein catalyzes the degradation of phenylalanine. Directional deletion of this gene can both block phenylalanine consumption and increase precursor supply, and upregulate... SCAB_82261 , txtR , txtA txtE Gene transcription, through a dual mechanism, increased thaxtomin A production by 110.5%. Simultaneously, the deletion did not affect strain growth, and gene restoration restored the original yield. The related methods are simple to operate and have stable effects, providing key technical support for the industrial production of thaxtomin A.

[0034] The above description is merely 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_82261 A method for increasing thaxtomin A production using genes, characterized by: First, genetic engineering was used to target Streptomyces scabies in Streptomyces scabies. SCAB_82261 Gene deletion was performed to obtain a high-yielding strain of *Streptomyces thaxtomin A* causing scabies, which was then used to produce thaxtomin A through fermentation; wherein, the... SCAB_82261 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

2.

2. The method according to claim 1, using *Streptomyces scabies* SCAB_82261 A method for increasing thaxtomin A production using genes, characterized by: The above in Streptomyces scabies SCAB_82261 In the gene deletion step, the specific Streptomyces scabies strain used was Streptomyces scabies strain 87.22, and the gene deletion was achieved through suicide plasmid pUCTSR and homologous recombination technology.

3. The method according to claim 1, using *Streptomyces scabies* SCAB_82261 A method for increasing thaxtomin A production using genes, characterized by: In the step of fermenting to produce thaxtomin A, the fermentation process conditions 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.

4. The method according to any one of claims 1 to 3 using *Streptomyces scabies* SCAB_82261 A method for increasing thaxtomin A production using genes, characterized by: The SCAB_82261 The gene encodes the phenylacetic acid-CoA ligase PaaK, which catalyzes the degradation of aromatic compounds, including phenylalanine; by deleting the [specific gene]... SCAB_82261 The gene blocks the degradation and metabolism of phenylalanine, increasing the accumulation of phenylalanine in the cell, and simultaneously upregulates the thaxtomin A biosynthesis gene cluster. txtR , txtA , txtE Increase the transcriptional level of genes and enhance the biosynthetic yield of thaxtomin A.

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