CN-121992047-A - Method for improving thaxtomin A yield through streptomyces scab SCAB _78491 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 scab SCAB _78491 gene, which comprises the steps of constructing a thaxtomin A high-yield strain through deleting a SCAB _78491 gene of Streptomyces scab through a genetic engineering way and fermenting the strain, wherein the sequence of the SCAB _78491 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2. The invention identifies SCAB _78491 gene in streptomyces scab as negative regulation gene for biological synthesis of thaxtomin A for the first time through screening, and obtains the thaxtomin A high-yield engineering strain by directionally deleting SCAB _78491 gene in streptomyces scab, thereby effectively improving the yield of thaxtomin A and providing technical support for industrial fermentation optimization.

Inventors

• LIU YINUO
• LIU JING
• Tang Youya
• LI ZHE
• YANG ENDONG

Assignees

• 安徽农业大学

Dates

Publication Date

20260508

Application Date

20260206

Claims (5)

1. 1. A method for improving the yield of thaxtomin A through Streptomyces scab SCAB _78491 gene is characterized in that the SCAB _78491 gene in Streptomyces scab is deleted through a genetic engineering way, a high-yield strain of thaxtomin A is constructed, the obtained high-yield strain is utilized for fermentation production of thaxtomin A, the nucleotide sequence of the SCAB _78491 gene is shown as SEQ ID NO.1, and the encoded amino acid sequence is shown as SEQ ID NO. 2.
2. 2. The method for increasing the yield of thaxtomin A by Streptomyces scab SCAB _78491 gene according to claim 1, wherein the genetic engineering approach uses suicide plasmid pUCTSR and homologous recombination technique to achieve deletion of SCAB _78491 gene in Streptomyces scab.
3. 3. The method for increasing the yield of thaxtomin a by the streptomyces scab SCAB _78491 gene according to claim 1, characterized in that the streptomyces scab is in particular the streptomyces scab 87.22 strain.
4. 4. The method for improving the yield of thaxtomin A by using Streptomyces scab SCAB _78491 gene according to claim 1, wherein the process conditions for producing thaxtomin A by fermentation are that spores of the thaxtomin A high-producing strain are inoculated into TSB seed medium, shake-cultured at 28℃and 220rpm for 2 days, and then transferred to OBB liquid fermentation medium, shake-cultured at 28℃and 220rpm for 7 days.
5. 5. The method for increasing the yield of thaxtomin A through Streptomyces scabus SCAB _78491 gene according to any one of claims 1 to 4, wherein the coding product of the SCAB _78491 gene is Lrp family transcription regulatory protein for negatively regulating the biosynthesis of thaxtomin A, and the negative regulation of thaxtomin A biosynthesis is released by deleting the SCAB _78491 gene to up-regulate the transcription level of txtR, txtA, txtE gene in the thaxtomin A biosynthesis gene cluster, thereby increasing the biosynthesis yield of thaxtomin A.

Description

Method for improving thaxtomin A yield through streptomyces scab SCAB _78491 gene Technical Field The invention relates to the technical field of genetic engineering, in particular to a method for improving the yield of thaxtomin A through Streptomyces scab SCAB-78491 genes. Background Streptomyces as a gram-positive filamentous bacterium with high G+C content has the capacity of biosynthesis of various secondary metabolites with complex structures and various functions, and the metabolites are widely applied to a plurality of fields such as medical health, animal husbandry, industry and agricultural herbicides. In streptomycete, synthetic genes of secondary metabolites are concentrated and distributed in genome in the form of gene clusters, and the expression activity of the synthetic gene clusters can be precisely regulated by various regulation genes, and the synthetic gene clusters mainly comprise three regulation modes of pathway specific regulation, multiple-effect regulation and global regulation. Therefore, the synthesis efficiency of the secondary metabolite of streptomycete can be effectively regulated and controlled by directionally modifying the regulating gene, the molecular mechanism of the secondary metabolism regulation is deeply analyzed, and the method has important promotion significance for constructing engineering strains with excellent performance by utilizing a genetic engineering technology and being applied to actual production. The invention is based on the thought, and aims to directionally modify the regulatory genes through a genetic engineering approach, so that the thaxtomin A high-yield strain is obtained, and the production requirement of the thaxtomin A is met. Thaxtomin a exhibits great potential for application in the field of agricultural herbicides. Compared with traditional chemical herbicides such as glyphosate, the thaxtomin A has more prominent herbicidal activity on broadleaf weeds and almost no harm to gramineous crops such as barley, rice, corn and the like, so how to increase the yield of the thaxtomin A has become a hot problem in current research. From the action mechanism, thaxtomin A can induce plant cell hypertrophy under nanomolar concentration, inhibit normal cell elongation, and further cause seedling growth retardation, and the core action target is to inhibit plant cellulose biosynthesis. From the synthetic point of view, the biosynthesis of thaxtomin A depends on a gene cluster consisting of 7 genes, of which 6 synthetic genes (txtA, txtB, txtC, txtD, txtE, txtH) are responsible for coding the corresponding biosynthetic enzymes and 1 regulatory gene txtR codes for the AraC/XylS family of transcriptional regulatory proteins which, by activating the expression of the txtA, txtB, txtC and txtD genes, initiate the biosynthesis process of thaxtomin A. Lrp (leucine response regulatory protein) family proteins are transcription regulatory factors widely existing in prokaryotes, play a key role in organisms, and can participate in regulating and controlling amino acid metabolism, central metabolism, energy metabolism, substance transport and other important physiological processes. At present, scientific researchers have carried out systematic research on the regulatory function of Lrp protein in antigen-producing bacteria, but in pathogenic streptomyces such as streptomyces scab, the regulatory function of Lrp protein and related molecular mechanisms still need to be further explored. Meanwhile, research on improving thaxtomin A yield in Streptomyces scab by means of regulatory genetic engineering is relatively deficient, and the existing report only involves indirectly improving txtR gene expression level by deleting regulatory factor gene cebR or SCAB _Lrp2, thereby improving thaxtomin A yield. The team of the invention discovers that SCAB _78491 gene codes for Lrp family protein based on bioinformatics analysis prediction of streptomyces scab genome sequence, and subsequent laboratory researches further prove that the yield of thaxtomin A is obviously improved after the streptomyces scab SCAB _78491 gene is deleted, so that SCAB _78491 is a negative regulatory factor involved in the biosynthesis of thaxtomin A. Accordingly, there is a need to establish an effective method for improving thaxtomin A yield by controlling Streptomyces scab SCAB _78491 gene. Disclosure of Invention The technical problem to be solved by the invention is to provide a method for improving the yield of thaxtomin A through streptomyces scab SCAB-78491 genes. The invention adopts the following technical scheme to solve the technical problems: A method for improving the output of thaxtomin A by using Streptomyces scab SCAB-78491 gene includes such steps as deleting SCAB-78491 gene from Streptomyces scab by genetic engineering, constructing the high-yield strain of thaxtomin A, and fermenting to obtain thaxtomin A by using the high-yield strain, wherein the nucleotide sequence of SCAB