CN116121163A

CN116121163A - Method for changing biosynthesis of thaxtomin A through transcription factor gene bldD in streptomyces scab

Info

Publication number: CN116121163A
Application number: CN202211713056.2A
Authority: CN
Inventors: 董胜男; 刘静; 杨恩东; 张部昌
Original assignee: Anhui Agricultural University AHAU; Anhui University
Current assignee: Anhui Agricultural University AHAU; Anhui University
Priority date: 2022-12-29
Filing date: 2022-12-29
Publication date: 2023-05-16

Abstract

The invention provides a method for changing thaxtomin A biosynthesis through a transcription factor gene bldD in streptomyces scab, and carrying out deletion or/and over-expression on the transcription factor gene bldD in the streptomyces scab through a genetic engineering approach, thereby obtaining a bldD gene deletion mutant strain/bldD gene over-expression strain; the nucleotide sequence of the bldD gene is shown as SEQ ID NO. 1. The modified strain obtained by the genetic engineering technology can reduce or increase biosynthesis of the secondary metabolite thaxtomin A of streptomyces scab, wherein, the mutant strain for reducing the biosynthesis of thaxtomin A is used for reducing scab and provides a new theoretical support for preventing and treating potato scab in agricultural production; mutants that increase thaxtomin a biosynthesis are useful as herbicides.

Description

Method for changing biosynthesis of thaxtomin A through transcription factor gene bldD in streptomyces scab

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for changing thaxtomin A biosynthesis through a transcription factor gene bldD in streptomyces scab.

Background

Streptomyces is a gram-positive filamentous bacterium with a G+C content of up to 70% to 74%, and more than 580 Streptomyces have been identified so far. Streptomyces produces a plurality of secondary metabolites with complex structures and various functions, and the metabolites are widely applied to medical health, animal husbandry, industry and agricultural herbicides. The synthetic genes of secondary metabolites in streptomyces are mostly located in the genome in clusters, and the synthetic gene clusters are regulated by various regulatory genes. At the genetic level, there are three levels of regulation of streptomycete secondary metabolism, respectively: pathway-specific regulation, multiple-effect regulation, and global regulation. The synthesis of secondary metabolite of streptomycete can be influenced through a series of genetic modification, and the research of the molecular mechanism of secondary metabolism regulation has great promotion effect on the application of ideal engineering strain obtained by using genetic engineering means to actual production. The invention aims to obtain a Streptomyces scab engineering strain by directionally changing genes through a genetic engineering way, which is used for preventing and treating potato scab in agricultural production.

Streptomyces scab is one of main pathogenic bacteria causing potato scab, and since the scab is generated, the damage to potatoes and the influence of the streptomyces scab on the whole potato industry are widely focused, and research on scab pathogen, pathogenic toxin, prevention and the like by researchers at home and abroad is gradually deepened, so that better research progress is achieved at present. Thaxtomin, which is produced by secondary metabolism of streptomyces scab, is a major causative agent of potato scab and acts as a cellulose synthesis inhibitor, causing cork-like lesions on potato tuber surfaces. Thaxtomins are a class of compounds with unique 2, 5-diketopiperazine structures that initiate scab by blocking cellulose synthesis during plant cell expansion and division. The scab disease not only seriously affects the yield and quality of potatoes, but also infects main crops such as radishes, beet, carrots and the like, and even comprises Chinese medicinal plants such as divaricate saposhnikovia root.

Thaxtomin a is the most metabolically active and structurally represented product of the thaxtomins family. the characteristic structural units of thaxtomin A are 4-nitroindole, which is a chemical structural unit unique to the thaxtomin family in all microbial sub-metabolites, and C-hydroxy-piperazine dione rings, which are present in many natural products with biological activity. The biosynthesis gene cluster (txt cluster) of Thaxtomin A is formed by sequentially arranging total 7 genes of txtC, txtH, txtB, txtA, txtR, txtE and txtD, and researches show that the biosynthesis of Thaxtomin A is regulated by transcription regulating factors such as transcription regulating factors TxtR and external BldD in the biosynthesis gene cluster.

BldD protein is a global transcription regulating factor which is researched to be mature in streptomycete, and can obviously regulate and control a plurality of important biological processes such as aerial hypha formation, spore production, antibiotic synthesis and the like of the streptomycete. bld the bald-type gene can regulate and control secondary metabolism and morphological differentiation of streptomycete. The bld family includes bldA, bldB, bldC, bldD, bldG, bldH and the like. Wherein the control mechanism of the bldD gene is studied more clearly. BldD can regulate 167 transcription units as a global regulator in Streptomyces coelicolor, for example, including regulatory genes that affect differentiation and regulatory genes that affect secondary metabolism. Based on bioinformatic predictions of the genomic sequence of streptomyces SCAB, the streptomyces SCAB scab_75171 gene is a bldD transcription regulatory family gene, and it was found by the present laboratory study that the streptomyces SCAB transcription factor gene bldD is a positive regulatory factor involved in thaxtomin a biosynthesis.

Accordingly, there is an urgent need for a method of altering thaxtomin A biosynthesis by Streptomyces scab transcription factor gene bldD.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for changing thaxtomin A biosynthesis through a transcription factor gene bldD in streptomyces scab.

The invention adopts the following technical scheme to solve the technical problems:

a method for changing thaxtomin A biosynthesis through transcription factor gene bldD in streptomyces scab comprises the steps of deleting or/and over-expressing transcription factor gene bldD in streptomyces scab through a genetic engineering way, so as to obtain bldD gene deletion mutant strain/bldD gene over-expression strain, and producing thaxtomin A by using the obtained strain; wherein, the nucleotide sequence of the bldD gene is shown as SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO. 2.

As one of the preferred modes of the present invention, the gene product of the bldD gene positively regulates the biosynthesis of thaxtomin A.

As a preferred embodiment of the present invention, the bldD gene deletion mutant is obtained by using Streptomyces scab 87.22 as an initial strain, and by suicide plasmid pUCTSR and homologous recombination technique. The Streptomyces scab 87.22 strain is a strain which is disclosed at present and obtained by the public, and is provided by China general microbiological culture collection center (CGMCC) 4.1765.

As one of the preferred modes of the present invention, the bldD gene deletion mutant reduces thaxtomin A biosynthesis for reducing scab.

As a preferred embodiment of the present invention, the bldD gene overexpression strain is obtained by introducing the vector pKC-bldD into an original strain starting from Streptomyces scab 87.22.

As one of the preferred modes of the present invention, the bldD gene overexpression strain is used for high-yield thaxtomin A, and is used as a herbicide.

Compared with the prior art, the invention has the advantages that:

the invention deletes the transcription regulation gene bldD in streptomyces scab by suicide plasmid pUCTSR and homologous recombination technology, thereby obtaining a gene deletion mutant strain with changed biosynthesis of thaxtomin A, and providing technical support for researching biosynthesis of secondary metabolite thaxtomin A of streptomyces scab. Wherein, when the bldD gene is knocked out in streptomyces scab 87.22, the yield of a secondary metabolite thaxtomin A in the ΔbldD mutant strain is greatly reduced; when the bldD gene is supplemented back in the ΔbldD mutant strain, the yield of thaxtomin A is recovered; from this, it was shown that Streptomyces scab BldD is a positive regulator involved in thaxtomin A biosynthesis. Meanwhile, the bldD gene is overexpressed in Streptomyces scab 87.22 by using the high copy plasmid pKC1139, and the yield of thaxtomin A is improved. These demonstrate that deletion and overexpression of the bldD gene can be used to targeted alter the biosynthetic yield of thaxtomin A in Streptomyces scab.

Drawings

FIG. 1 is a diagram showing the positional information of bldD and adjacent genes on a chromosome;

FIG. 2 is a schematic diagram of construction of deletion mutants using suicide plasmid pUCTSR and homologous recombination technique;

FIG. 3 is a schematic diagram showing PCR verification of a bacterial solution of a ΔbldD deletion mutant strain (in the figure, a bldD gene on a chromosome of Streptomyces scab is replaced by a thiostrepton resistance gene tsr, and after 303bp of the bldD gene is replaced by 1360bp of the tsr gene, a PCR amplified band of a positive clone is 1558bp;M,5000bp DNA Marker);

FIG. 4 is an HPLC analysis of thaxtomin A production in fermentation products of original strain 87.22 and ΔbldD related strain;

FIG. 5 is a transcriptional analysis of thaxtomin A biosynthesis-related genes in ΔbldD deletion mutants (in the figures, a graph shows thaxtomin A biosynthesis gene cluster genes txtA, txtE and txtR 24h transcriptional analysis, and b graph shows thaxtomin A biosynthesis gene cluster genes txtA, txtE and txtR 48h transcriptional analysis);

FIG. 6 is an EMSA analysis of the promoter region of the genes involved in BldD protein and thaxtomin A biosynthesis;

FIG. 7 is an HPLC analysis of thaxtomin A yield in fermentation products of original strain 87.22 and bldD gene overexpression strain.

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.

The strains and plasmids used in the examples below are shown in Table 1, and the primer sequences used are shown in Table 2.

Coli used in the following examples was cultured in liquid LB medium at 37℃or on solid LB plates supplemented with 2% agar. Streptomyces scab is cultured in TSB medium at 28℃or on ISP-2 solid medium. The culture medium used for fermentation is oat bran liquid (OBB) culture medium, and the preparation method comprises the following steps: (1) Adding 20g of oat bran powder into 1L of water, boiling for about 20min, and boiling to paste; (2) Filtering with 3 layers of gauze, collecting supernatant, and fixing volume to 1L; (3) Adjusting the pH of the culture medium to 7.2-7.4, subpackaging into conical flasks, and sterilizing at 121 ℃ for 20min.

TABLE 1 bacterial species and plasmids used in the examples of the invention

/>

TABLE 2 primers used in the examples of the present invention

/>

/>

Example 1

bldD gene related information:

the bldD and the positional information of adjacent genes on the chromosome are shown in FIG. 1.

The nucleotide sequence of the bldD gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

Example 2

Construction of Δbldd deletion mutant strain (see fig. 2):

the primer sequences in this experiment are shown in Table 2. The genome DNA of streptomyces scab is used as a template, bldD-up-F/R and bldD-down-F/R are used as primers, a 1500bp homologous fragment on the upstream and downstream of a bldD gene part is amplified by PCR, a target fragment is recovered, hindIII, xbaI, kpnI and EcoRI are used for double digestion of the upstream and downstream fragment, agarose gel electrophoresis is used for recovering the target fragment, and then the target fragment is cloned to pUCTSR plasmid to obtain recombinant plasmid pUCTSR- ΔbldD, and double digestion verification is carried out on pUCTSR- ΔbldD.

The constructed recombinant plasmid was transferred into E.coli ET12567 (pUZ 8002), the pUCTSR- ΔbldD plasmid which was verified to be correct was transformed into Streptomyces scab by using the homologous recombination technique, positive mutant strains were screened by resistance to thiostrepton, mycelium PCR analysis was performed on the selected strains by using the primer bldD-C-F/R, and the positive mutant strains which were verified to be successful were named ΔbldD (FIG. 3).

Example 3

Construction of reverting and overexpressing strains:

the genome DNA of Streptomyces scab is used as template, the primer bldD-C-F/R is used to amplify complete bldD gene segment, and EcoRI and HindIII enzyme cutting sites are introduced separately. The PCR product was recovered by agarose gel, cloned into pBluescript II SK (+) and sequenced. The recombinant plasmid pKC1139-bldD is obtained by double digestion of EcoRI and HindIII after the correct sequence, and is connected to an episomal vector pKC1139, the digestion result proves that the recombinant plasmid construction is successful, the correct pKC1139-bldD plasmid and the empty pKC1139 plasmid are transformed into competent cells of escherichia coli ET12567 (pUZ 8002), the pKC-bldD and the empty pKC1139 are respectively introduced into wild-type streptomyces scab 87.22 and delta bldD in deletion mutant strains by the same method, after the conjugation transfer, the resistance zygote of the apramycin is obtained by screening, and after the PCR verification by Apr primers, the correct over-expression strain 87.22/pKC-bldD and the empty control strain 87.22/pKC1139, and the complementing strain delta bldD/pKC1139 are confirmed.

Example 4

Detection of Streptomyces scab fermentation product:

inoculating TSB seed bottle cultured Streptomyces scab and related strain into fermentation culture medium, and culturing at 28 deg.C for 6-7 days at rotation speed of 160 rpm/min. After fermentation of Streptomyces scab and related strains is completed, the fermentation broth is extracted with 20mL of ethyl acetate, the supernatant is obtained after centrifugation, the supernatant is distilled by a rotary evaporator, the distilled sample is dissolved in 2mL of methanol, and the filtered solution is directly used for detecting the thaxtomin A content after a 0.22 mu m organic filter membrane is used for filtering. thaxtomin a content assay (HPLC method): the high performance liquid chromatography column adopts Agilent C18 column, 4.6X105 mm,5.0 μm; the flow rate is 0.5mL/min; the mobile phase adopts 65% formic acid and 35% acetonitrile; the detection wavelength is 380nm; the column temperature is 40 ℃; the sample loading amount was 10. Mu.L.

Example 5

Purification of proteins and EMSA analysis:

the genome DNA of streptomyces scab is used as a template, bldD-28a-F/R is used as a primer, a complete bldD gene fragment is amplified by PCR, a target fragment is recovered, ndeI and HindIII are used for double enzyme digestion, the target fragment is recovered by agarose gel electrophoresis, and then the target fragment is cloned to pET28a plasmid, so that recombinant plasmid pET28a-bldD is obtained.

The plasmid is transformed into competent E.coli BL21 to obtain a protein expression host BL21/pET-bldD. Transferring a protein expression host BL21/pET-bldD into LB seeds containing Kana antibiotics according to a proportion of 1%, transferring the protein expression host BL21/pET-bldD into 50mL of LB+Kana culture medium according to a proportion of 2% in the next day, culturing in an oscillating way until the OD value is 0.5-0.8, adding IPTG with a final concentration of 0.5mmol/L, and oscillating for 20h at 16 ℃; and (4) taking proper bacterial liquid for SDS-PAGE analysis, and primarily detecting whether the protein is expressed successfully.

Crushing the successfully expressed protein by using an ultrasonic crusher, transferring the crushed thalli into a centrifuge tube, pre-cooling the centrifuge at 4 ℃, centrifuging at 12000rpm for 30min, and separating supernatant and sediment; the supernatant solution was passed through a 0.22 μm aqueous filter, and then purified by adding the supernatant solution to a nickel column in which the supernatant solution was equilibrated, and the protein eluted with 500mmol/L imidazole was the target protein.

The txtin A toxin synthesis genes txtA, txtE and txtR are used for PCR amplification of the promoter regions of the txtA, txtE and txtR genes by using txtA-EMSA-F/R, txtE-EMSA-F/R and txtR-EMSA-F/R as primers and Streptomyces scab genome as templates, and the fragments are used in protein EMSA experiments and are respectively named PtxtA, ptxtE, ptxtR probes in sequence, and the corresponding fragments are recovered by agarose gel electrophoresis. The EMSA system combining BldD protein and DNA is 20 mu L, after being fully mixed, the mixture is incubated in a constant temperature box at 30 ℃ for 10-20 min, and the whole system is spotted into 6% active PAGE gel holes for electrophoresis.

Example 6

Transcription analysis:

the fermentation broths of 24h and 48h of the original strains Streptomyces scab 87.22 and ΔbldD deletion mutant strain are used for extracting total RNA of bacteria by using a kit, and are reversely transcribed into cDNA, and the transcription level is detected by using a fluorescence quantitative PCR instrument.

Analysis of results:

1. deletion and overexpression of the bldD gene can alter the production of thaxtomin A

The initial strain Streptomyces scab 87.22, the delta bldD deletion mutant strain, the anaplerotic strain delta bldD/pKC-bldD and empty load control strain delta bldD/pKC1139 thereof, the over-expression strain 87.22/pKC-bldD and empty load control strain 87.22/pKC1139 thereof are fermented and cultured, and the yield of thaxtomin A in each strain is quantitatively analyzed after the fermentation is finished. HPLC detection of thaxtomin A (FIG. 4) showed a significant decrease in yield of ΔbldD deletion mutant compared to starting strain 87.22; the production of thaxtomin A in the anaplerotic strain ΔbldD/pKC-bldD was substantially restored, indicating that the decrease in thaxtomin A in the ΔbldD deletion mutant was indeed caused by a mutation in bldD: whereas the overexpression strain 87.22/pKC-bldD had an increased thaxtomin A yield compared to the empty control 87.22/pKC 1139. These demonstrate that deletion and overexpression of the bldD gene can be used to targeted alter the biosynthetic yield of thaxtomin A in Streptomyces scab.

2. BldD positively regulates transcript levels of thaxtomin A biosynthetic genes

qRT-PCR analysis was performed on the transcript level of gene txtA, txtE, txtR in the S.scab thaxtomin A synthetic gene cluster with 16sRNA as an internal reference. As a result, it was confirmed that the expression level of the above-mentioned genes was significantly reduced in the ΔbldD deletion mutant at 24h and 48h compared with that of the original strain 87.22 (FIG. 5A, B), indicating that the deletion of bldD could down-regulate the transcription level of thaxtomin A biosynthesis gene in Streptomyces scab, confirming that Streptomyces scab BldD affects thaxtomin A biosynthesis by positively regulating the transcription level of the related genes.

3. Promoter of BldD binding thaxtomin A biosynthesis gene

The promoter region of txtA, txtE, txtR gene was amplified by PCR using Streptomyces scabus genome as template and each probe was incubated with purified BldD fusion protein, respectively, and EMSA analysis showed that when BldD protein was added, the protein and each probe DNA showed obvious in vitro binding action, respectively, compared with the control without protein added (FIG. 6), indicating that BldD was able to directly regulate transcription of genes related to thaxtomin A biosynthesis.

4. Overexpression of the bldD Gene increases the yield of thaxtomin A

The original strain 87.22 and the overexpressing strain 87.22/pKC-bldD were fermented in the same manner as described above and then subjected to HPLC analysis (FIG. 7), which showed an increase in thaxtomin A content in the overexpressing strain compared to the starting strain 87.22.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. A method for changing thaxtomin A biosynthesis through transcription factor gene bldD in streptomyces scab is characterized in that deletion or/and over-expression is carried out on the transcription factor gene bldD in streptomyces scab through a genetic engineering approach, so as to obtain a bldD gene deletion mutant strain/bldD gene over-expression strain, and the obtained strain is used for producing thaxtomin A; wherein, the nucleotide sequence of the bldD gene is shown as SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown as SEQ ID NO. 2.

2. The method of changing thaxtomin a biosynthesis by a transcription factor gene bld in streptomyces scab according to claim 1, wherein the gene product of the bldD gene positively regulates thaxtomin a biosynthesis.

3. The method for altering thaxtomin A biosynthesis by bldT, a transcription factor gene in Streptomyces scab according to claim 1, wherein the bldD gene deletion mutant is a strain starting from Streptomyces scab 87.22, and the gene deletion is achieved by suicide plasmid pUCTSR and homologous recombination technique.

4. The method of altering thaxtomin a biosynthesis by a transcription factor gene bld in streptomyces scab according to claim 1, wherein the bldD gene deletion mutant reduces thaxtomin a biosynthesis for reducing scab.

5. The method for altering the biosynthesis of thaxtomin A by bldE, a transcription factor gene in Streptomyces scab according to claim 1, wherein the bldE gene overexpression strain is obtained by introducing vector pKC-bldD into an original strain using Streptomyces scab 87.22 as an original strain.

6. The method of altering thaxtomin a biosynthesis by bld, a transcription factor gene in streptomyces scab according to claim 1, characterized in that the bld gene overexpression strain is used for high yield thaxtomin a for use as herbicide.