CN117587054A - Lysobacter enzymogenes mutant strain capable of producing HSAF at high yield and construction method and application thereof - Google Patents
Lysobacter enzymogenes mutant strain capable of producing HSAF at high yield and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a lysobacter enzymogenes mutant strain with high HSAF yield, and a construction method and application thereof, belonging to the technical field of microorganisms; according to the research of the invention, csrA has global regulation and control effect on the biocontrol capability of the lysobacter enzymogenes OH11, and the knockout or silencing of the coding gene of the CsrA protein in the lysobacter enzymogenes OH11 can improve the yield of the antifungal oomycete active substance HSAF in the lysobacter enzymogenes OH11 to be nearly 3 times; the invention carries out deletion mutation on the coding gene of CsrA protein in the lysobacter enzymogenes OH11 through genetic modification to obtain the lysobacter enzymogenes mutant strain; the construction method of the lysobacter enzymogenes mutant strain is simple, does not need additional special fermentation conditions, can obviously improve the yield of the lysobacter enzymogenes HSAF, and has good practical value.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a lysobacter enzymogenes mutant strain capable of producing HSAF at high yield, a construction method and application thereof.
Background
HSAF (heat stable antifungal factor, thermostable antifungal factor) is a secondary metabolite produced primarily by lysobacter enzymogenes (Lysobacter enzymogenes) that is capable of broad-spectrum antagonism of filamentous fungi and oomycetes. At present, HSAF has been demonstrated to have remarkable control ability against various plant diseases caused by fungi or oomycetes, such as pear rot and wheat scab. Meanwhile, HSAF has also been demonstrated to have excellent antifungal properties for the treatment of diseases caused by fungi, such as: fungal keratitis, fungal skin infections, and the like. HSAF has the following advantages over other antifungal agents currently on the market: (1) the HSAF has a novel chemical structure, belongs to a polycyclic tetramic macrolactam (polycyclic tetramate macrolactams, poTeMs) compound, and contains a tetramic acid structural unit and a 5/5/6 tricyclic system; (2) HSAF has a unique bacteriostatic mechanism that inhibits the growth of filamentous fungal sphingolipids by interfering with their synthesis; (3) HSAF has high safety and is harmless to plants and mammals. Thus, HSAF has the potential to be developed as a novel microbial source antibiotic.
Lysobacter enzymogenes OH11 is a representative strain that naturally produces HSAF, whereas the yield of HSAF is very low for wild-type OH11 strain, and is only 1.8 μg/mL, which limits the development process of HSAF. Although the yield of HSAF in the lysobacter enzymogenes OH11 can be improved to 572.95 mug/mL by optimizing fermentation conditions and the like at present, the yield requirement of industrial production cannot be met. Therefore, in addition to optimization of fermentation conditions, modifications to the lysobacter enzymogenes OH11 strain are required to enhance its ability to synthesize HSAF.
At present, some methods for improving the HSAF-producing ability of OH11 strains by genetic engineering are developed successively, such as by knocking out 10 c-di-GMP synthesis related genes in OH11 or by knocking out or silencing iron-responsive transcription factor Fur genes in OH11, but the methods have the problems that the strain construction workload is large or special fermentation conditions need to be provided for mutant strains. Therefore, there is a need to develop a high HSAF-producing lysobacter enzymogenes which is simple in construction method and does not require special fermentation conditions.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a lysobacter enzymogenes mutant strain with high HSAF yield, and a construction method and application thereof; according to the research of the invention, csrA has global regulation and control effect on the biocontrol capability of the lysobacter enzymogenes OH11, and the knockout or silencing of the coding gene of the CsrA protein in the lysobacter enzymogenes OH11 can improve the yield of the antifungal oomycete active substance HSAF in the lysobacter enzymogenes OH11 to be nearly 3 times; the invention carries out deletion mutation on the coding gene of CsrA protein in the lysobacter enzymogenes OH11 through genetic modification to obtain the lysobacter enzymogenes mutant strain; the construction method of the lysobacter enzymogenes mutant strain is simple, does not need additional special fermentation conditions, can obviously improve the yield of the lysobacter enzymogenes HSAF, and has good practical value.
In order to achieve the technical effects, the invention adopts the following technical means:
the invention firstly provides application of an RNA binding protein CsrA in constructing lysobacter enzymogenes mutant strain with high HSAF yield.
Preferably, the nucleotide sequence encoding the CsrA is shown in SEQ ID No. 1.
Preferably, the use comprises silencing or knocking out the nucleotide sequence encoding CsrA.
The invention also provides a construction method of the lysobacter enzymogenes mutant strain for high production of HSAF, which specifically comprises the following steps: the nucleotide sequence encoding CsrA in the lysobacter enzymogenes is knocked out or silenced.
Preferably, the method comprises: amplifying an upstream fragment and a downstream fragment of the csrA gene, respectively treating the upstream fragment and the downstream fragment by using restriction enzymes, and then connecting the treated upstream fragment and the treated downstream fragment to a vector to obtain a recombinant vector;
transferring the recombinant vector into lysobacter enzymogenes, and carrying out homologous recombination to obtain the high-yield HSAF lysobacter enzymogenes mutant strain.
Preferably, the lysobacter enzymogenes is lysobacter enzymogenes OH11.
Preferably, the vector comprises pEX18Gm.
The invention also provides a recombinant vector comprising an upstream fragment and a downstream fragment of the csrA gene.
The invention also provides the lysobacter enzymogenes mutant strain with high HSAF yield constructed by the method.
The invention also provides the application of the high-HSAF-yield lysobacter enzymogenes mutant strain or the high-HSAF-yield lysobacter enzymogenes mutant strain prepared by the method in producing the HSAF.
Compared with the prior art, the invention has the beneficial effects that:
the invention carries out deletion mutation on the coding gene of CsrA protein in the lysobacter enzymogenes OH11 through genetic modification to obtain the lysobacter enzymogenes mutant strain with high HSAF yield; the yield of the antifungal oomycete active substance HSAF in the high-yield HSAF-producing lysobacter enzymogenes mutant strain is improved to be nearly 3 times compared with that of the wild-type HSAF-producing lysobacter enzymogenes OH11.
The invention also discovers that the mutant growth rate and the twitching motility capacity of the lysobacter enzymogenes with high yield of HSAF after csrA knockout are influenced, which indicates that the csrA has global regulation and control function on the biocontrol capacity of the lysobacter enzymogenes OH11, and the lysobacter enzymogenes with high yield of HSAF can be used for researching the biocontrol mechanism of the lysobacter enzymogenes OH11.
Compared with the method for improving the HSAF yield by genetic modification of OH11 in the prior art, the method for constructing the lysobacter enzymogenes mutant strain for producing the HSAF in high yield is simpler, does not need additional special fermentation conditions compared with a wild strain, can obviously improve the HSAF yield of the lysobacter enzymogenes, and has good popularization and application values.
Drawings
FIG. 1 is a graph showing HSAF production by a mutant strain of Acetobacter enzymogenes (csrA in the figure) and a wild-type OH11 strain (WT in the figure) which produce HSAF; wherein a is a chromatogram at 318nm, B is a plot of HSAF yield, p <0.001.
FIG. 2 shows the growth curves of the high HSAF-producing lysobacter enzymogenes mutant (csrA in the figure) and the wild-type OH11 strain (WT in the figure).
FIG. 3 shows the twitching motility of high HSAF-producing lysobacter enzymogenes mutant (csrA in the figure) and wild-type OH11 strain (WT in the figure); in the figure, p <0.01; * P <0.001.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto. In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The sources of the reagents used, the trade names and the components of the reagents are shown when the reagents appear for the first time, and the reagents which are the same as the sources shown for the first time are not specially indicated; the reagents, materials, etc. involved are commercially available without any particular explanation. In the following examples, the media LB and 10% TSB media used are all commonly used in the art and are formulated as a universal formulation.
The original strain adopted by the following implementation, namely, the lysobacter enzymogenes OH11 is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms (including No. 2007) in the year 03 and 19: CGMCC No.1978; the sucrose-sensitive suicide plasmid pEX18Gm is commonly used in the art and is commercially available.
Example 1: construction of lysobacter enzymogenes mutant strain with high HSAF yield
In the embodiment, the lysobacter enzymogenes mutant strain with high HSAF yield is constructed by taking the lysobacter enzymogenes OH11 as an initial strain, and the specific steps are as follows:
(1) The upstream and downstream fragments Up-csrA (SEQ ID No. 2) and Dw-csrA (SEQ ID No. 3) of the gene csrA (SEQ ID No. 1) were obtained by PCR amplification using the primers Up-csrA-F/R, dw-csrA-F/R.
Wherein, the primer sequences are as follows:
Up-csrA-F：GATGTGCTGCAAGGCGATTACGTTTCGATTTTTCGCACTT(SEQ ID No.4)；
Up-csrA-R：CCGAATCGTCCGTCAGGATCAGCATTGAAA(SEQ ID No.5)；
Dw-csrA-F：GATCCTGACGGACGATTCGGGCAAACAC(SEQ ID No.6)；
Dw-csrA-R：CCTCTAGAGTCGACCTGCAGGCCTGAGTGCAATTATGACG(SEQ IDNo.7)。
SEQ ID No.1：
ATGCTGATCCTGACGCGTCGCGTCGGCGAAACCCTGATGATCGGCGACTCGGTGACGGTCACCGTGCTCGGCGTCAAGGGCAACCAGGTGCGTATCGGTATTACCGCACCGAAAGATGTCGCGGTACATCGCGAGGAGATTTACCAGCGCATTCAGCGCGGCGAGGATGCGTCGGACGATTCGGGCAAACACACGGGCGGCTGA
SEQ ID No.2：
GATGTGCTGCAAGGCGATTACGTTTCGATTTTTCGCACTTCAACCCGCTGACCGACGCCGAACTCGCCGAACTCGAGCGCCAGGTCAACCGCGACGTGCGCGGCAACTACGAGGCCGAAGTCCGCCAGATGGGCATGCAGGAAGCCCTGGACTTCGGCGCCATGGCGCTGTTCGGCGAGAAGTACGGCGACCGCGTGCGCGTGTTGCGCATGGGCGAGGCCTCGACCGAGCTGTGCGGCGGCACCCACGTGTCGCGCACCGGCGACATCGGTCTGTTCAAGATCGTCGCCGAAGGCGGCGTGCAGGCCGGCGTGCGCCGGATCGAGGCGGTGACCGGGCAGGGCGCGCTGGACTACGTGGCCGAAGAGGAAGCGCGCCTGGAGCAGGCCGCGCGCCTGCTCGGCGGCAACCCGGCCGACGTCGCCGACAAGCTGCGCGCGTTGCTGGACCGGCAGAAGAAGCTCGAGCGCGAGCTCGAATCGATCAAGGCCAAGGCCGCCTCCGGCGCCGCGGCCGACCTGGCCGCCTCGGCGGCGCAGATCGGTCCGGTCAAGGTCGTGGCCACCCGGCTGGAAGGTTTCGACGCCAAGGCGCTGCGCGAGGCGGTCGACCGTCTCAAGCAACAGCTTGGCGATGCGGTGATCGTGCTGGCGGGCGCCAGCGACGGTAAGGCGGCCTTGGTCGCCGGAGTGAACGGAAGCGCGGCTGGCAAGGTCAAGGCGGGGGAACTCATCTCGCACGTGGCCGGTCAGATCAACGGTAAAGGCGGCGGCCGACCGGACATGGCTCAGGGCGGCGGCGACGACGGCCCGGCCCTGGCCCAGGCTCTTGCGGGCGTCGCGCGATGGGTTGAGAGTAAGCTTGTCTGAATGGCGAACAGTGACTGCTATCGCTTGCGACCGGGTTTGACCGGTCGCTAAGATGGTTCACGTTTGTGTACAAATTGAAGCATTCTTCCATATGGAAAGAATGCCAACGCCGGTGCCCCGGGTGCCGGCTCGAGGAGGTTTTCAATGCTGATCCTGACGGACGATTCGG
SEQ ID No.3：
GATCCTGACGGACGATTCGGGCAAACACACGGGCGGCTGAGGAACAGGTCGCCTGAGAGCATCGCCGCGAATGTTGCGGCGGATGTTGCTGTAAATCGGGCGAGCCGTTATGATTGCCGCCCGCATCGCTTGATAGATGCGGAGAGTTGCCCGAGTGGCCGAAGGGGCTCCCCTGCTAAGGGAGTATAGGGTCAAAAACTCTATCGAGGGTTCGAATCCCTCACTCTCCGCCAGTTGTACCGAAGCCGCCTTCGGCGGCTTTTCTTTCGCCGCACCGGCCATGCGACACGGCCGGTACGACGAAGGGGTTTCACCGGAAATGTTGACGGAAAGTCGTTTGTCCGTCATACTTTTCCAACTAAGCGCCCGTAGCTCAGCTGGATAGAGCACCAGGCTACGAACTTGGGGGTCGGAGGTTCGAATCCTTCCGGGCGCGCCATATTGAGAAGCCGGCAATCGCGAGATTGCCGGCTTTTTCTTTTGCCTGGGTTTTTGTCGGCGCTGCCGCGAATCGCCTGCGGTTTTGGGTTTTGCAGGCCAACTCGCGCGAGCGGAGTTCGTCGTTTCGGGTGGTGACGCGACGAGGTACGAGGCATTGAGCGACGAACCGACGAAGCCCCGCAGCATCGAAGCGCCCGGGCCTTGCCGTGCATTGCGATAGCGTCGTCGCCGTGCCCGCCGCCGAAGCGACGCAGCGACGTGGGAGGGCCTTCAGGCCCGACGCCCTGCGATCGGATCGCCGCCGCCGAAATGCAGCGCAGCAAGCGCATATGCAAACAAGCCTGGACGCCGGCCCGTGGCTGCGCCGACGCGATGTTTTCCCGATGTGTCGCGATAGGGCATCGACGTCGCGATGACGCTTCCCGTCGCGCGAATAACACGTCATAATTGCACTCAGGCCTGCAGGTCGACTCTAGAGG。
(2) The sucrose-sensitive suicide plasmid pEX18Gm is subjected to linearization treatment through PCR to obtain a linear vector pEX18Gm, then DNA fragments Up-csrA and Dw-csrA are seamlessly integrated into the linear vector pEX18Gm by using Gibson assembly technology, and a tool plasmid csrA_Up-Dw-pEX18Gm is constructed.
The tool plasmid csrA_Up-Dw-pex18gm is a homologous recombination tool plasmid, does not contain a complete DNA fragment of csrA, but contains an upstream fragment and a downstream fragment of csrA, and can be used as a homology arm to carry out homologous recombination with an OH11 genome of the lysobacter enzymogenes, so that the mutant bacterium of the lysobacter enzymogenes with high yield of HSAF is obtained.
(3) The tool plasmid csrA_Up-Dw-pex18gm was placed in the wild type strain of lysobacter enzymogenes OH11, and the strain transformed into the tool plasmid was plated on LB agar plates containing 150. Mu.g/mL gentamicin, and cultured at 28℃for 48 hours. The single-clone dot-dash line was picked up and cultured on LB agar medium containing 15% sucrose without sodium chloride at 28℃for 48 hours, and the strain in which the secondary recombination occurred was selected.
The monoclonal is selected and respectively spotted on LB and LB culture plates containing 150 mug/mL gentamicin, and the spots growing only on LB agar culture plates are selected for PCR identification and sequencing. Sequencing shows that the sequence at the csrA of the strain is mutated into the sequence shown as SEQ ID No.8, so that the unmarked knockout of the csrA gene is successfully realized, and the high-yield HSAF lysobacter enzymogenes mutant strain is obtained.
SEQ ID No.8：
ATGCTGATCCTGACGGACGATTCGGGCAAACACACGGGCGGCTGA。
Example 2: determination of HSAF production
In this example, the ability of producing hsa by using the high-yield hsa-producing lysobacter enzymogenes mutant strain constructed in example 1 was tested by using High Performance Liquid Chromatography (HPLC) with OH11 wild type strain as a control group, and the specific method is as follows:
the single colonies of OH11 wild type strain and high-yield HSAF lysobacter enzymogenes mutant strain are respectively cultured in LB liquid medium at 28 ℃ in a shaking way for overnight, then 500 mu L of bacterial liquid cultured overnight is respectively transferred into 25mL of 10% TSB culture liquid, and the bacterial liquid is placed in a shaking table at 28 ℃ for culturing for 24 hours. After the completion of the culture, 4mL of each bacterial liquid was taken, 16. Mu.L of concentrated hydrochloric acid was added thereto and mixed well, and then 4mL of ethyl acetate was added thereto and gently shaken for 1 hour. After the oscillation is finished, standing for layering, taking 2mL of the upper layer solution into a centrifuge tube, drying by a fume hood, adding 200 mu L of methanol for dissolution, and then taking 20 mu L of sample for HPLC detection.
Wherein the mobile phase is acetonitrile and water, the chromatographic column is Agilent SB-C18 (250 mm×4.6mm), the detection wavelength is 318nm, and the detection result is shown in FIG. 1.
FIG. 1 is a graph showing the HSAF yield of a mutant strain of lysobacter enzymogenes (csrA in the figure) and a wild-type OH11 strain (WT in the figure), wherein under the same conditions, the HSAF yield of the mutant strain of lysobacter enzymogenes with high HSAF yield constructed by the present invention is 3 times that of the wild-type OH11 strain.
Example 3: determination of growth rate of lysobacter enzymogenes mutant strain for high HSAF production
In this example, the growth rate of the high HSAF-producing lysobacter enzymogenes mutant strain was tested using the OH11 wild type strain as a control group, and the specific steps are as follows:
and respectively picking a single bacterial colony of the wild type OH11 of the lysobacter enzymogenes and a single bacterial colony of the mutant lysobacter enzymogenes with high yield of HSAF, and shake culturing the single bacterial colony in an LB liquid culture medium at 28 ℃ for overnight. Inoculating the bacterial liquid into a new 10% TSB liquid culture medium according to the proportion of 1%, and continuing 28 ℃ shake culture. 150. Mu.L of bacterial liquid is taken out at different time points respectively, and OD is measured by a spectrophotometer 600 Values were recorded and a complete growth graph was finally formed, and the measurement results are shown in fig. 2.
FIG. 2 shows growth curves of high HSAF-producing lysobacter enzymogenes mutant strain (csrA in the figure) and wild-type OH11 strain (WT in the figure), and it can be seen from FIG. 2 that the growth rate of high HSAF-producing lysobacter enzymogenes mutant strain is significantly slower in the retention adaptation phase than that of wild-type OH11.
Example 4: determination of scratch movement ability of lysobacter enzymogenes mutant bacteria with high HSAF yield
In this embodiment, the OH11 wild strain is used as a control group to test the ability of the high-yield HSAF lysobacter enzymogenes mutant strain to twitch motion, and the specific test method is as follows:
the single colony of the lysobacter enzymogenes OH11 and the single colony of the lysobacter enzymogenes mutant strain of the high-yield HSAF are respectively dipped by using a sterile toothpick, and the bottoms of the flat-plate culture medium containing 1% agar and LB and 10% TSB are penetrated for static culture at 28 ℃. After 48h and 72h of incubation, the plaque diameter at the bottom of the medium was counted and the results are shown in FIG. 3.
FIG. 3 shows the twitching motility capacity of the high-yield HSAF-producing lysobacter enzymogenes mutant strain (csrA in the figure) and the wild-type OH11 strain (WT in the figure), and as can be seen from FIG. 3, the diameter of the twitching motility ring of the high-yield HSAF-producing lysobacter enzymogenes mutant strain is smaller than that of the wild-type strain, which indicates that the twitching motility capacity is weaker than that of the wild-type strain.
As can be seen by combining fig. 2 and fig. 3, the growth rate of the high-yield HSAF lysobacter enzymogenes mutant strain in the detention adaptation period is reduced, and the scratch ability is obviously weakened, which indicates that the biocontrol ability of the mutant is changed in multiple ways, and the mutant can be used for researching the biocontrol mechanism of the lysobacter enzymogenes OH11.
In conclusion, the coding gene of CsrA protein in the lysobacter enzymogenes OH11 is subjected to deletion mutation through genetic modification to obtain the lysobacter enzymogenes mutant strain with high HSAF yield; the yield of the antifungal oomycete active substance HSAF in the high-yield HSAF-producing lysobacter enzymogenes mutant strain is improved to nearly 3 times compared with the yield of the high-yield HSAF-producing lysobacter enzymogenes OH11. In addition, the construction scheme of the lysobacter enzymogenes mutant strain for high production of HSAF is simpler, does not need additional special fermentation conditions compared with a wild strain, can obviously improve the yield of the lysobacter enzymogenes HSAF, and has good popularization and application values.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above-described embodiments, and any obvious modifications, substitutions or variations that can be made by one skilled in the art without departing from the spirit of the present invention are within the scope of the present invention.
Claims (10)
- Application of RNA binding protein CsrA in constructing high-HSAF-producing lysobacter enzymogenes mutant bacteria.
- 2. The use according to claim 1, characterized in that the nucleotide sequence encoding CsrA is shown in SEQ ID No. 1.
- 3. The use according to claim 1, wherein the use comprises silencing or knocking out the nucleotide sequence encoding CsrA.
- 4. A method for constructing a mutant bacterium of lysobacter enzymogenes with high HSAF yield, comprising the steps of: the nucleotide sequence encoding CsrA in the lysobacter enzymogenes is knocked out or silenced.
- 5. The method for constructing a mutant bacterium of lysobacter enzymogenes which produces HSAF in high yield according to claim 4, wherein the method comprises: amplifying an upstream fragment and a downstream fragment of the csrA gene, respectively treating the upstream fragment and the downstream fragment by using restriction enzymes, and then connecting the treated upstream fragment and the treated downstream fragment to a vector to obtain a recombinant vector;transferring the recombinant vector into lysobacter enzymogenes, and carrying out homologous recombination to obtain the high-yield HSAF lysobacter enzymogenes mutant strain.
- 6. The method for constructing a mutant bacterium of lysobacter enzymogenes having high HSAF yield according to claim 5, wherein said lysobacter enzymogenes is lysobacter enzymogenes OH11.
- 7. The method of constructing a high HSAF producing lysobacter enzymogenes mutant strain according to claim 5, wherein the vector comprises pEX18Gm.
- 8. A recombinant vector comprising an upstream fragment and a downstream fragment of a csrA gene.
- 9. The lysobacter enzymogenes mutant strain of high HSAF yield constructed by the method of any one of claims 4 to 7.
- 10. The use of the high HSAF producing lysobacter enzymogenes mutant strain of claim 9, or the high HSAF producing lysobacter enzymogenes mutant strain constructed by the method of any one of claims 4 to 7, in producing HSAF.
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