A PhlH protein mutant and its application in high-yield 2,4-diacetylphloroglucinol

Abstract

The invention discloses a PhlH protein mutant and its application in high-yield 2,4-diacetyl phloroglucinol, and relates to the field of genetic engineering technology. The mutant is prepared by mutating the 144th amino acid of the PhlH protein sequence from lysine to alanine. The present invention is to reintegrate the phlH point mutation gene phlH / K144A in the phlH gene deletion strain of Pseudomonas fluorescens 2P24 to weaken the negative feedback regulation of PhlH protein on 2,4-DAPG biosynthesis, and significantly improve the synthesis level of 2,4-diacetyl phloroglucinol. The present invention can be further applied to industrial production to change the current status of low yield of 2,4-DAPG.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a PhlH protein mutant and application thereof in high-yield 2,4-diacetylphloroglucinol. Background Art

[0002] Pseudomonas bacteria, which are beneficial to plants, actively cultivate plant roots and produce many secondary metabolites. Among them, 2,4-DAPG is an important phenolic secondary metabolite. Due to its broad-spectrum antibacterial activity, it helps to inhibit wheat take-all disease, tomato black root disease, wheat root rot and other plant diseases. Therefore, it is widely used in agricultural production and is an important biocontrol factor for fluorescent Pseudomonas.

[0003] The molecular structure of 2,4-DAPG is simple. It can be iteratively condensed into 3,5-diketopimelate by three molecules of malonyl-CoA, then decarboxylated and cyclized to form phloroglucinol, and then acetylated twice to form MAPG and DAPG, respectively. This synthetic pathway is controlled by the highly conserved phl biosynthetic gene cluster in Pseudomonas, which includes structural genes (phlACBD), efflux genes (phlE), degradation genes (phlG) and regulatory genes (phlF and phlH). The regulatory genes phlF and phlH from the TetR family play a regulatory role by encoding two pathway-specific regulatory factors. The transcription factor PhlH encoded by the phlH gene can form a dimer and bind to the upstream of the degradation gene phlG, and strongly inhibit its expression, thereby preventing the degradation of 2,4-DAPG mediated by PhlG. At the same time, 2,4-DAPG can also interact with PhlH, separate the PhlH-DNA complex, and then release the inhibitory effect on phlG.

[0004] At present, the synthesis methods of 2,4-DAPG include chemical synthesis and biosynthesis. Among them, chemical synthesis has harsh conditions, high cost and large pollution, while biosynthesis mainly utilizes the biosynthesis of Pseudomonas fluorescens cells themselves. This method is highly respected because of its low condition requirements, low pollution and renewable synthetic raw materials, and is the mainstream approach for the synthesis of 2,4-DAPG. However, due to the presence of factors such as degradation genes (phlG), 2,4-DAPG is easily degraded in vivo, so that the yield of 2,4-DAPG synthesized by existing technical means is low, and it is difficult to meet industrial production. Therefore, there is an urgent need in the art to construct an engineering strain with high yield of 2,4-DAPG, improve the industrial yield of 2,4-DAPG, so that it can be widely used in actual production and actively play an antibacterial activity. Summary of the invention

[0005] The purpose of the present invention is to provide an engineered strain with high 2,4-DAPG yield to address the problem of low 2,4-DAPG yield caused by negative feedback regulation of phlH, thereby increasing the industrial yield of 2,4-DAPG and enabling it to be widely used in actual production and actively exert antibacterial activity.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The invention provides a PhlH protein mutant capable of producing high 2,4-DAPG. The mutant is prepared by mutating the 144th amino acid of the PhlH protein sequence from lysine to alanine. The PhlH protein is derived from Pseudomonas fluorescens 2P24. The PhlH protein mutant is recorded as PhlH / K144A.

[0008] A further improvement is that the amino acid sequence of the PhlH protein is shown as SEQ ID NO.2, the nucleotide sequence of its encoding gene phlh is shown as SEQ ID NO.1, and the amino acid sequence of the mutant PhlH / K144A is shown as SEQ ID NO.2.

[0009] The present invention also provides a phlh / K144A2 gene, which is used to encode the above-mentioned PhlH protein mutant, and its nucleotide sequence is shown in SEQ ID NO.3.

[0010] The present invention also provides a recombinant plasmid, which contains the phlh / K144A2 gene.

[0011] A further improvement is that the recombinant plasmid is a pBBR5pemIK plasmid.

[0012] The present invention also provides a genetically engineered bacterium with high 2,4-DAPG production, wherein the genetically engineered bacterium comprises the recombinant plasmid or the phlh / K144A2 gene is integrated into the genome.

[0013] A further improvement is that the genetically engineered bacteria is Escherichia coli DH5α or Pseudomonas fluorescen 2P24.

[0014] The present invention also provides an application of the genetically engineered bacteria in increasing the yield of 2,4-DAPG.

[0015] The principle of the present invention is that 2,4-DAPG is one of the many secondary metabolites produced by Pseudomonas fluorescens, and its content can be kept dynamically stable in Pseudomonas fluorescens. The negative feedback of phlH plays a vital role here, and is also the main reason for the limited biosynthesis yield of 2,4-DAPG. The present invention reduces the affinity of phlH for 2,4-DAPG through site-directed mutagenesis, thereby obtaining a mutant strain with higher 2,4-DAPG yield, which has high application value.

[0016] The present invention has the following beneficial effects:

[0017] Site-directed mutations are produced in amino acid residues of the PhlH protein that recognizes and binds to 2,4-DAPG to weaken the negative feedback regulation of the PhlH protein on the biosynthesis of 2,4-DAPG, thereby increasing the yield of 2,4-DAPG.

[0018] The following abbreviations or abbreviations are used in the present invention:

[0019] Negative feedback: A phenomenon in which a metabolic reaction is inhibited by its reaction product is called negative feedback;

[0020] Pseudomonasfluorescens 2P24 is a biocontrol strain isolated by Professor Zhang Liqun of China Agricultural University from the soil infected with wheat take-all disease in Shandong. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of pK18mobSacB-phlH vector;

[0022] Figure 2 Schematic diagram of pBBR5pemIK::phlH vector;

[0023] Figure 3 pBBR5pemIK::phlH / K144A Schematic diagram of the vector;

[0024] Figure 4 This is the high performance liquid chromatography detection chart of the standard product 2,4-DAPG;

[0025] Figure 5 This is a high performance liquid chromatography detection chart of wild-type 2,4-DAPG;

[0026] Figure 6 This is the 2,4-DAPG HPLC detection diagram of the knockout strain ΔphlH;

[0027] Figure 7 This is the 2,4-DAPG HPLC detection diagram of the complemented empty vector strain ΔphlH / pBBR5pemIK;

[0028] Figure 8 To complement the mutant strain ΔphlH / pBBR5pemIK::phlH K144A 2,4-DAPG high performance liquid chromatography detection chart;

[0029] Fig. 9 The figure is a comparison chart of the final production of 2,4-DAPG of each strain. DETAILED DESCRIPTION

[0030] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0031] 1. Materials

[0032] The materials, reagents, instruments and methods used in the following methods are conventional in the art and can be obtained through commercial channels unless otherwise specified.

[0033] (1) The enzyme reagents used were purchased from Thermo Company, and the kits used for plasmid extraction and DNA fragment recovery were purchased from Vazyme Company. The corresponding operation steps were carried out according to the product instructions.

[0034] (2) The knockout plasmid pK18mobsacB carries the nptII (kanamycin resistance gene) resistance marker and the sacB selectable marker.

[0035] (3) The pBBR5pemIK plasmid carries the broad-spectrum replicon pBBR1, which can replicate autonomously in a variety of host cells. The pBBR1 replicon is a medium-to-low copy replicon that carries the pemI / K anti-endotoxin gene, which can improve the stability of the plasmid in the host cell; it carries a gentamicin resistance marker.

[0036] (4) Culture medium formula:

[0037] 1) Shake flask seed culture medium

[0038] LB liquid culture medium: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121°C for 20 min, the concentration of each component is the final concentration in the culture medium.

[0039] 2) Shake flask fermentation medium

[0040] KB liquid medium: Tryptone 20g / L, Glycerol 10mL / L, MgSO 4 .7H2 O 1.5g / L, K 2 HPO 4 1.5g / L, pH 7.2, sterilized at 121℃ for 20min. The concentrations of each component are the final concentrations in the culture medium.

[0041] 2. Methods

[0042] 2.1 Construction of a recombinant strain that produces high-yield 2,4-diacetylphloroglucinol

[0043] 2.1.1 Construction of phlH knockout plasmid

[0044] Using Pseudomonas fluorescens 2P24 genomic DNA as a template, the nucleotide sequence of Pseudomonas fluorescens phlH is shown in SEQ ID NO.4, and the upstream and downstream homology arms of PhlH were obtained by PCR amplification:

[0045] The sequence of the upstream homology arm forward primer PhlH-UP-F is: 5'-CGGAATTCGCCGTGGTCCTGTTGACCCACG-3', as shown in SEQ ID NO.7;

[0046] The sequence of the reverse primer PhlH-UP-R is: 5'-GCTCTAGAATGAGCTATGCCCTTGGCGGCC-3', as shown in SEQ ID NO.8;

[0047] The sequence of the downstream homology arm forward primer PhlH-DOWN-F is: 5'-AATCTAGAGTTGGCCAACCCCTGCTCGGCA-3', as shown in SEQ ID NO.9

[0048] The sequence of the reverse primer Ph1H-DOWN-R is: 5'-CCCAAGCTTTTGCTGTATTTTTTCGACAGC-3', as shown in SEQ ID NO.10.

[0049] The cloned upstream homology arm was double-digested with EcoRⅠ and XbalⅠ, and the downstream homology arm was double-digested with XbalⅠ and HindⅢ. After the vector pK18mobSacB was double-digested with EcoRⅠ and HindⅢ, the upstream and downstream homology arm fragments and the vector pK18mobSacB fragment after enzyme digestion were respectively recovered using a recovery kit, and the three fragments were connected using a ligase. The connection product was transformed into E.coliDH5α, and the positive clones were screened to obtain the recombinant plasmid pK18mobSacB-phlH (such as Figure 1The nucleotide sequences of its upstream and downstream homology arms are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0050] 2.1.2 Preparation of 2P24 strain lacking phlH

[0051] The successfully recombined knockout vector pK18mobSacB-phlH was transformed into the competent cells of the donor strain S17-1 by transformation, and it was inoculated into LB medium (containing Kana antibiotics) as the donor strain of the gene knockout vector and cultured overnight at 37°C. At the same time, the strain planned to be gene knocked out, Pseudomonas fluorescens 2P24, was cultured at 28°C. After the above-mentioned donor and recipient strains grew up, the bacteria were collected by low-speed centrifugation (to prevent the bacteria from rupturing), and then washed 2-3 times with fresh antibiotic-free culture medium, and then resuspended with a certain volume of fresh culture medium. Next, the S17-1 strain with the suicide plasmid was used as the vector supply strain and the 2P24 vector receiving strain was cultured according to a certain The ratio of the ratio was mixed, and the mixed strain was dropped onto the non-antibiotic KB solid culture medium and cultured at 28°C for 8-10h. At this time, the gene knockout vector would be joined from the donor strain S17-1 to the recipient strain 2P24. Then the plaque was scratched with a sterilized gun tip and inoculated into a fresh liquid culture medium with double resistance (the suicide plasmid itself has Kana resistance and the vector recipient bacteria 2P24 itself has Amp resistance) for culture. After the strain grew up, the bacterial solution was diluted according to a certain dilution ratio and evenly spread on the Kana+Amp double antibiotic plate to force it to undergo the first homologous recombination under antibiotic pressure. After 20h, a single clone was picked and the single exchange strain that had integrated the suicide vector into its genome was screened by PCR.

[0052] The recipient strain that had been verified to have undergone a single exchange was then cultured in an antibiotic-free culture medium for 10 hours, then diluted in an appropriate ratio and evenly coated with a solid KB culture medium containing 10% sucrose, forcing it to undergo a second homologous recombination under the lethal pressure of sucrose. The recipient strain that had undergone a double exchange was picked, and the bacterial liquid PCR method was used to verify the target gene knockout strain, thereby obtaining the deletion strain ΔphlH.

[0053] 2.1.3 Construction of recombinant plasmid pBBR5pemIK-phlH

[0054] The phlH gene fragment was obtained by PCR amplification using Pseudomonas fluorescens 2P24 genomic DNA as a template;

[0055] The sequence of the forward primer phlH-F is: 5'-CGGGGTACCATGGACAATGCCATTGGCAA-3', as shown in SEQ ID NO.11;

[0056] The sequence of the reverse primer phlH-R is: 5'-CCCAAGCTTTCAGCTTGCAGCATCGTGCG-3', as shown in SEQ ID NO.12;

[0057] The phlH gene and vector pBBR5pemIK were double-digested with KpnⅠ and HindⅢ using a gel recovery kit, and the recovered enzyme-digested fragments and the vector were separated by T 4 DNA ligase was used for ligation, and the ligation product was heat-shock transformed into E. coli DH5α cloning strain, and positive single clones were screened to obtain the recombinant plasmid pBBR5pemIK-phlH. Figure 2 shown.

[0058] 2.1.4 Recombinant plasmid pBBR5pemIK-phlH K144A Construction

[0059] The recombinant plasmid pBBR5pemIK-phlH constructed above was used as a DNA template to construct pBBR5pemIK-phlH / K144A in which lysine 144 was mutated to alanine. The amino acid sequence of Pseudomonas fluorescens PhlH is shown in SEQ ID NO.1. The plasmid was point mutated by PCR. High-fidelity DNA polymerase was used to perform point mutation by circular PCR to obtain the target fragment.

[0060] Forward primer phlH K144A -F sequence: 5'-GCTCAAGGAGCAATCGTTTCCCGCGGCACACATCC-3', as shown in SEQ ID NO.13;

[0061] Reverse primer phlH K144A The -R sequence is: 5'-AGCAAGCCACGCAGGATGTGTGCCGCGGGAAAC-3', as shown in SEQ ID NO.14.

[0062] Then, the methylated original template in the recovered product was digested with the fast-cutting enzyme DpnⅠ to obtain the recombinant plasmid pBBR5pemIK-phlH K144A (like Figure 3 The nucleotide sequence of the mutated phlH gene is shown in SEQ ID NO.3, and the amino acid sequence of the PhlH protein mutant is shown in SEQ ID NO.2.

[0063] 2.1.5 Construction of recombinant strains

[0064] The recombinant plasmid pBBR5pemIK-phlH constructed above wasK144A The recombinant strain was obtained by introducing the phlH gene-deficient strain of Pseudomonas fluorescens 2P24 through conjugation transfer. The recombinant strain is a phlH gene-complemented mutant strain of Pseudomonas fluorescens 2P24, and the specific method is as follows:

[0065] A. construct the complement plasmid pBBR5pemIK-phlH K144A Heat shock transformation was performed into the E. coli S17-1λpir clone strain, and positive single clones were selected and placed in LB liquid medium, and cultured at 37°C and 200 rpm until OD600 = about 0.6-0.8; at the same time, single clones of the phlH gene deletion strain of Pseudomonas fluorescens 2P24 were selected and placed in LB medium, and cultured at 28°C and 200 rpm for about 16 minutes;

[0066] B. After the cultured Escherichia coli and Pseudomonas aeruginosa are co-cultured on 0.45 μm filter paper at a certain ratio (1:1, 1:2), they are screened using a double resistance plate, and positive single clones are picked to obtain recombinant strains.

[0067] 2.2 Vial fermentation test of recombinant strains

[0068] In this example, two groups of experiments were conducted, each group was independently conducted three times and the average value was taken to verify that the recombinant strain of the present invention has the effect of increasing the yield of 2,4-diacetylphloroglucinol.

[0069] Control group: wild-type strain—Pseudomonas fluorescens 2P24

[0070] Experimental groups: recombinant strains—knockout strain ΔphlH of Pseudomonas fluorescens 2P24, complemented empty vector strain ΔphlH / p BBR5pemIK, complemented mutant strain ΔphlH / pBBR5pemIK::phlH K144A .

[0071] 2.2.1 Vial fermentation experiment

[0072] Single clones of the wild-type strain and the recombinant strain were picked and inoculated into 50 mL shake flasks containing 20 mL LB, respectively. Appropriate antibiotics were added as needed, and the mixture was shaken and cultured at 28°C and 200 rpm for about 16 h until the OD 600 When the concentration reached 1.0, the inoculum was transferred to a 250 mL shake flask containing 50 mL KB at a 5% inoculum volume, and appropriate antibiotics were added as needed. The fermentation was continued at 28°C and 200 rpm with shaking until the end.

[0073] 2.2.2 Determination of 2,4-DAPG content in each strain by HPLC

[0074] Sampling is performed in time periods, and 1 mL of fermentation liquid is taken to a 1.5 mL centrifuge tube every 12 hours, and then 0.1 mL of fermentation liquid is taken to an ELISA plate, and the real-time OD is measured by an ELISA instrument, which is used as the basis for evaluating the credibility of the results later. The present invention takes fermentation liquid samples of five time periods of 12, 24, 36, 48, and 60, and after centrifugation at 12000 rpm for 10 minutes, the supernatant is transferred to a new 1.5 mL centrifuge tube, filtered with a 0.22 μm organic filter head, and transferred to an HPLC injection bottle for inspection, and the remaining bacterial precipitate can be dried with a freeze dryer for dry weight determination.

[0075] Then, a standard product was prepared by weighing 10 mg of 2,4-DAPG and dissolving it in 1 mL of ethanol to prepare a 10 g / L stock solution; the solution was then gradiently diluted to 20 mg / L, 50 mg / L, 100 mg / L, and 500 mg / L.

[0076] Subsequently, the 2,4-DAPG content was detected by HPLC (mobile phase: phase A is acetonitrile; phase B is ddH2O; chromatographic column: WondaSilC18-WRcolumn (5μm, 4.6x150mm); detection conditions: phase A 55%, phase B 45%, flow rate 1mL / min, column temperature 30°C, detection wavelength 270nm, detection time 5min / sample, 2,4-DAPG retention time is 2.3min). The results are as follows Figure 4-8 as shown.

[0077] According to the above example operation steps, the vial fermentation data are as follows:

[0078] The average 2,4-DAPG production of the control group, namely the wild-type strain of Pseudomonas fluorescens 2P24, was 81.679 mg / L

[0079] The average 2,4-DAPG production of the knockout strain ΔphlH of Pseudomonas fluorescens 2P24 in the experimental group was 15.261 mg / L; the average 2,4-DAPG production of the complemented empty strain ΔphlH / pBBR5pemIK was 8.416 mg / L; the complemented mutant strain ΔphlH / pBBR5pemIK::phlH K144A The 2,4-DAPG production of the three groups of samples were 266.496 mg / L, 321.534 mg / L, and 267.910 mg / L, respectively. K144A The average 2,4-DAPG yield of the sample was 285.313 mg / L. Fig. 9 shown.

[0080] The Pseudomonas fluorescens 2P24 complemented mutant strain ΔphlH / pBBR5pemIK::phlH of the present invention K144AThe 2,4-DAPG production of the strain was increased by 249% compared with that of the wild-type strain 2P24.

[0081] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A PhlH protein mutant PhlH / K144A, It is characterized in that The mutant is prepared by mutating the 144th amino acid of the PhlH protein sequence from lysine to alanine. The PhlH protein sequence is shown in SEQ ID NO.1, and the amino acid sequence of the mutant is shown in SEQ ID NO.

2.

2. a phlh / K144A gene, It is characterized in that The phlh / K144A2 gene encodes the mutant according to claim 1.

3. A phlh / K144A gene according to claim 2, It is characterized in that The nucleotide sequence of the phlh / K144A encoding gene is shown in SEQ ID NO.

3.

4. A recombinant plasmid, It is characterized in that The recombinant plasmid contains the phlh / K144A encoding gene as claimed in claim 3.

5. A recombinant plasmid according to claim 4, It is characterized in that The recombinant plasmid is pBBR5pemIK plasmid.

6. A genetically engineered bacterium with high yield of 2,4-DAPG, It is characterized in that The genetically engineered bacteria comprises the recombinant plasmid as described in any one of claims 4-5 or the genome comprises the phlh / K144A2 encoding gene as described in any one of claims 2-3.

7. A genetically engineered bacterium according to claim 6, It is characterized in that The genetically engineered bacteria are Escherichia coli DH5α or Pseudomonas fluorescen 2P24.

8. Use of the PhlH protein mutant PhlH / K144A as claimed in claim 1 in increasing 2,4-DAPG production.

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