A genetically engineered bacterium for producing phenoxazinone hydroxylated derivatives and a method for constructing the same

Abstract

The present application relates to the technical field of genetic engineering, and provides a genetically engineered bacterium for producing phenazine hydroxylated derivatives and a construction method thereof.The method is as follows: by constructing a recombinant plasmid of acyl-CoA dehydrogenase gene sgpK from Streptomyces griseus P510, the recombinant plasmid is introduced into Pseudomonas chlororaphis P3ΔH or wild-type Pseudomonas chlororaphis P3 for heterologous expression, and high-efficiency synthesis of hydroxylated PCA derivatives is realized.The hydroxylated PCA derivatives in the product are identified, and the derivatives have excellent bacteriostatic effect and can also be used as precursors for synthesis of rare medicines.The genetically engineered bacterium constructed in the present application has a 9-hydroxyphenazine-1-carboxylic acid (9-OH-PCA) yield of 470 mg / L, and has great industrial application potential and economic value.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic engineering technology, specifically to a genetically engineered bacterium that produces phenazine hydroxylated derivatives and its construction method. Background Technology

[0002] Phenazine derivatives are important natural compounds and a vital resource for drug development. Modifying and altering their structures is a crucial method for creating novel compounds. Changes in the core ring modification groups of phenazine compounds (such as alterations in the type, position, and number of functional groups) typically affect their redox potential and solubility, thus endowing them with different physicochemical properties and biological activities. 9-OH-PCA is a hydroxylated phenazine derivative with broad-spectrum antibacterial activity. Phenazine derivatives are mainly derived from chemical and biosynthetic methods. Traditional chemical synthesis methods have many drawbacks, such as numerous reaction steps, harsh reaction conditions, poor substitution selectivity, and the accumulation of toxic byproducts; while biosynthesis offers advantages such as high efficiency, environmental friendliness, and cost-effectiveness.

[0003] Heterologous expression biosynthesis is a novel method for expanding the diversity of natural products, developed in recent years with the advancement of biological technologies. Using microorganisms as "cell factories," heterologous expression biosynthesis optimizes endogenous metabolic pathways and rationally introduces exogenous synthetic pathways through gene deletion, gene substitution, gene insertion, and gene rearrangement, thereby synthesizing novel "non-natural" natural products, playing a crucial role in new drug development. *Pseudomonas aeruginosa* P3 is a safe and harmless biocontrol strain with a highly efficient phenazine synthesis pathway, rapid reproduction capacity, and good robustness, making it an ideal "cell factory" for synthesizing phenazine compounds. Therefore, based on P3's efficient phenazine synthesis pathway, and guided by the structure of the active compound, the biosynthesis of hydroxylated phenazine derivatives can be effectively achieved through the specific catalytic reaction of biological enzymes. Summary of the Invention

[0004] The purpose of this invention is to provide a genetically engineered bacterium that produces hydroxylated phenazine derivatives and a method for constructing the same. This invention guides the biosynthesis of hydroxylated phenazine derivatives through the catalysis of specific functional enzymes, guided by the structures of lead and target compounds.

[0005] The technical solution of the present invention is as follows:

[0006] A genetically engineered bacterium that produces high levels of phenazine hydroxylated derivatives was prepared by heterologously expressing the sgpK gene in *Pseudomonas aeruginosa*. The nucleotide sequence of the sgpK gene is shown in SEQ ID NO. 5.

[0007] The sgpK gene is derived from Streptomyces griseoluteus P510.

[0008] The *Pseudomonas aeruginosa* strain is selected from *Pseudomonas aeruginosa* P3 or *Pseudomonas aeruginosa* P3ΔH.

[0009] The method for constructing the genetically engineered bacteria includes the following steps:

[0010] S1. Extract the sgpK gene from Streptomyces griseoluteus P510;

[0011] S2. The pBBR1MCS2 plasmid was double-digested with QuickCut Dpn I and QuickCut Dpn II.

[0012] S3. The double-digested pBBR1MCS2 plasmid is ligated with the sgpK gene fragment to obtain the pBBR-sgpK recombinant plasmid.

[0013] S4. Transform the pBBR-sgpK recombinant plasmid into Pseudomonas aeruginosa to obtain the genetically engineered bacteria.

[0014] In step S1, the primers for extracting the sgpK gene include sgpK-F and sgpK-R, wherein the nucleotide sequence of sgpK-F is shown in SEQ ID NO.1; and the nucleotide sequence of sgpK-R is shown in SEQ ID NO.2.

[0015] The recombinant plasmid was transferred into Pseudomonas aeruginosa by electroporation.

[0016] Furthermore, the present invention also provides a recombinant plasmid containing a site-directed mutant sgpK-L155K gene, the nucleotide sequence of which is shown in SEQ ID NO.6.

[0017] The site-directed mutation sgpK-L155K gene is obtained by single-point mutation of LEU155 in the SgpK gene to LYS155, resulting in SgpK-LYS155.

[0018] Furthermore, the present invention also provides a genetically engineered bacterium that produces high levels of phenazine hydroxylated derivatives. The genetically engineered bacterium is prepared by heterologously expressing the site-directed mutant gene sgpK-L155K of the sgpK gene in Pseudomonas aeruginosa. The nucleotide sequence of the sgpK-L155K gene is shown in SEQ ID NO.6.

[0019] Furthermore, the present invention also provides a method for constructing a genetically engineered bacterium that produces high levels of phenazine hydroxylated derivatives, comprising the following steps:

[0020] Step 1: Extract the sgpK gene from Streptomyces griseoluteus P510;

[0021] Step 2: Single-point mutation of LEU155 in the SgpK gene to LYS155 to obtain the SgpK-LYS155 fragment, and amplify the SgpK-LYS155 fragment.

[0022] Step 3: Double digestion of pBBR-sgpK plasmid with QuickCut Dpn I and QuickCut Dpn II;

[0023] Step 4: Ligate the double-digested pBBR-sgpK plasmid with the SgpK-LYS155 fragment to obtain the pBBR-sgpKL155K recombinant plasmid.

[0024] Step 5: Transform the pBBR-sgpKL155K recombinant plasmid into Pseudomonas aeruginosa to obtain the genetically engineered bacteria.

[0025] Step 4, the construction method of the pBBR-sgpK plasmid includes the following steps:

[0026] S1. Extract the sgpK gene from Streptomyces griseoluteus P510;

[0027] S2. The pBBR1MCS2 plasmid was double-digested with QuickCut Dpn I and QuickCut Dpn II.

[0028] S3. The double-digested pBBR1MCS2 plasmid is ligated with the sgpK gene fragment to obtain the pBBR-sgpK recombinant plasmid.

[0029] In step 2, the primers used to amplify the SgpK-LYS155 fragment include L155K-F and L155K-R; the nucleotide sequence of L155K-F is shown in SEQ ID NO.3; and the nucleotide sequence of L155K-R is shown in SEQ ID NO.4.

[0030] In step 5, the *Pseudomonas chlororaphis* is *P. chlororaphis* P3ΔH.

[0031] This invention:

[0032] 1. By reviewing the literature, we explored the structures of phenazine derivatives whose biosynthetic pathways are currently unknown, mainly including 9-OH-PCA.

[0033] 2. Selection of genes and design of biosynthetic pathways for phenazine derivatives: Based on the structural characteristics of the aforementioned phenazine derivatives, PCA was identified as the precursor compound for this invention. Furthermore, through gene function analysis, the corresponding biocatalytic enzyme was determined. First, this invention identified the acyl-CoA dehydrogenase family protein SgpK from *Streptomyces glaucus* P510, which can catalyze the hydroxylation of the C9 position of the phenazine ring. Therefore, based on the compound's structure and the identified enzyme function, we selected SgpK to construct the 9-OH-PCA biosynthetic pathway.

[0034] 3. Construction of recombinant strains containing phenazine derivatives. After amplifying all the selected genes, construct the corresponding gene replacement plasmids, and then insert them into the selected locations in the strain genome using a binding transfer method to obtain a genetically engineered *Pseudomonas aeruginosa* strain that synthesizes phenazine derivatives.

[0035] 4. This invention constructs an overexpression plasmid of the acyl-CoA dehydrogenase gene sgpK from *Streptomyces glaucus* P510 and a recombinant plasmid of sgpK, which are then heterologously expressed in *Pseudomonas aeruginosa* P3ΔH and wild-type *Pseudomonas aeruginosa* P3, respectively, achieving efficient synthesis of hydroxylated PCA derivatives. The hydroxylated PCA derivatives in the products were identified, and these derivatives exhibit excellent antibacterial effects and can also serve as precursors for the synthesis of rare drugs. The genetically engineered bacterium constructed in this invention achieved a yield of 470 mg / L of 9-hydroxyphenazine-1-carboxylic acid (9-OH-PCA), demonstrating significant potential for industrial application and economic value. Attached Figure Description

[0036] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0037] Figure 1 It is a phenazine derivative structure; phenazine-1-carboxylic acid (PCA): R1 is -COOH, R2 is -H; 9-hydroxyphenazine-1-carboxylic acid (9-OH-PCA): R1 is -COOH, R2 is -OH.

[0038] Figure 2 Design diagram for the biosynthesis of phenazine derivatives; HPLC detection and yield diagram of N;

[0039] Figure 3 The HPLC detection results for 9-OH-PCA;

[0040] Figure 4 Comparison of binding energies for SgpK enzyme mutants;

[0041] Figure 5Comparison of the interactions between SgpK enzyme and L155K mutant with PCA molecules;

[0042] Figure 6 Yield of 9-OH-PCA by SgpK enzyme mutant strain during fermentation;

[0043] Figure 7 The 13C-NMR spectrum of 9-OH-PCA (DMSO, 400MHz). Detailed Implementation

[0044] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0045] like Figure 1 The structures of phenazine derivatives are shown; phenazine-1-carboxylic acid (PCA): R1 is -COOH, R2 is -H; 9-hydroxyphenazine-1-carboxylic acid (9-OH-PCA): R1 is -COOH, R2 is -OH;

[0046] Figure 2 Design diagram for the biosynthesis of phenazine derivatives; HPLC detection and yield diagram of N.

[0047] The strains, plasmids, and sequences involved in the following examples are as follows:

[0048] Table 1. Strains involved in this application

[0049]

[0050] Table 2. Plasmids involved in this application

[0051]

[0052]

[0053] Table 3 Primers involved in this application

[0054]

[0055] Table 4 Gene sequences involved in this application

[0056]

[0057]

[0058]

[0059] Example 1: Construction of engineered Pseudomonas aeruginosa strain for synthesizing PCA derivatives

[0060] Preparation methods for culture media and antibiotics:

[0061] LB liquid medium (1L): 10g tryptone, 5g yeast extract, 5g sodium chloride, adjust pH to 7.2, add 15g agar powder to solid medium (LB plate).

[0062] 15% sucrose solid medium (sucrose plate) (1L): yeast powder 5g, tryptone 10g, sucrose 150g, agar powder 15g.

[0063] YS (Yeast Starch Agar II) liquid medium (1L): 8g soluble starch, 2g yeast extract, adjust pH to 7.3; solid medium (YS plate) with 15g agar powder added.

[0064] Preparation of kanamycin solution: Weigh 0.5g of kanamycin powder and dissolve it in 10mL of sterile water. Filter the solution through a 0.22μm sterile filter to remove bacteria, then dispense it into individual containers and store it at -20℃.

[0065] Preparation of ampicillin solution: Weigh 1g of ampicillin sodium salt powder and dissolve it in 10mL of sterile water. Filter the solution through a 0.22μm sterile filter to remove bacteria, then dispense and store at -20℃.

[0066] 1. Construction of recombinant plasmid pBBR-sgpK

[0067] 1-1. Genome extraction from S. griseoluteus P510

[0068] Step 1: Cultivation of Streptomyces griseoluteus P510: Solid culture: Streak strain P510 was streaked onto YS solid medium and cultured at 28℃ for 4 days; Liquid culture: P510 spores were picked up with a sterile inoculation loop and inoculated into an Erlenmeyer flask containing 60ml of YS liquid medium (containing 10g of glass beads), and cultured with shaking at 200rpm and 28℃.

[0069] Step 2: Using the TIANamp Bacteria DNA Kit (TIANGEN), centrifuge to collect 0.1g of Streptomyces glaucus P510 precipitate, add lysozyme solution to a final concentration of 20μg / μL, and incubate in a 37℃ metal bath for 1-2 hours (for Gram-negative bacteria, only add 200μL of GA buffer and mix well); then add 20μL of Proteinase K and mix gently; add 220μL of buffer GB, mix gently by pipetting, and incubate at 70℃ for 10 minutes; add anhydrous ethanol and mix, then pour into a tube containing an adsorption column, centrifuge, and discard the liquid; then wash twice with PW buffer (containing alcohol), and incubate at room temperature for 10 minutes to allow the alcohol to evaporate completely; finally, add 50μL of preheated ddH2O to the membrane of the adsorption column, centrifuge at 12,000 rpm for 3 minutes, and freeze for later use.

[0070] 1-2. Amplification of the sgpK gene

[0071] The PrimeSTAR Max DNA Polymerase kit (Shanghai Haojia Technology Development Co., Ltd.) was used, and the corresponding primers were synthesized by Shanghai Ruimian Biotechnology Co., Ltd. The PCR reaction system is shown in Table 1-1.

[0072] Table 1-1 PCR amplification system (30 μL)

[0073]

[0074] 1-3, double digestion of pBBR1MCS2 plasmid

[0075] Using the QuickCut Dpn endonuclease kit (Shanghai Haojia Technology Development Co., Ltd.), the pBBR1MCS2 plasmid vector was digested at 37℃ for 2 hours according to the double digestion system in Table 1-2.

[0076] Table 1-2 Plasmid double enzyme digestion system (50 μL)

[0077]

[0078]

[0079] 1-4. Gel electrophoresis and gel recovery of PCR products and double enzyme digestion products

[0080] Using the HiPure Gel Pure DNA Mini Kit (Shanghai Chengyan Biotechnology Co., Ltd.), the PCR products from steps 1-2 and the double-digested plasmids from steps 1-3 were subjected to gel electrophoresis. Under blue light, the gel containing the target fragments (sgpK gene fragment and digested pBBR1MCS2 plasmid fragment) was excised and placed in a centrifuge tube. Solution B sol was added and the mixture was incubated at 50°C until completely dissolved. The solution was then transferred to an adsorption column, centrifuged, and the filtrate was discarded. Wash twice with WashSolution (containing alcohol), centrifuged again, and the residual alcohol was dried with a hairdryer.

[0081] Add 30 μL of preheated ddH2O to the center of the membrane, centrifuge to obtain the recovered DNA solution, and store it in a -20°C freezer for subsequent ligation steps.

[0082] 1-5. Seamless ligation of pBBR1MCS2 plasmid with the sgpK gene to create pBBR-sgpK plasmid.

[0083] Using the Seamless Cloning and Assembly Kit (Beijing Adley Biotechnology Co., Ltd.), the recovered linear vector (double-digested pBBR1MCS2 plasmid) was ligated with the sgpK gene fragment at 50℃ for 40 min according to the ligation system in Table 1-3.

[0084] Table 1-3 Connection systems (10 μL)

[0085]

[0086] 1-6 The pBBR-sgpK plasmid was introduced into Dh5a cells to complete the construction.

[0087] Preparation of Dh5a competent cells:

[0088] (1) Pick a single colony of DH5α into a vial containing LB liquid medium and incubate at 37°C with shaking for 12 h;

[0089] (2) Transfer 1% inoculum into an Erlenmeyer flask containing 60 mL LB medium and incubate at 200 rpm and 16 °C until the OD600 is 0.4-0.5;

[0090] (3) Let stand on ice for 10 min, centrifuge at 4,000 rpm at low temperature (4℃) for 15 min, and discard the supernatant;

[0091] (4) Add 50 mL of low-temperature sterile 0.1 M CaCl2 and resuspend the bacterial cells;

[0092] (5) Place in an ice bath for 15 min, centrifuge at 4,000 rpm at low temperature (4℃) for 10 min, discard the supernatant; repeat the operation once;

[0093] (6) Add 1 mL of low-temperature sterile 0.1M CaCl2 and 15% glycerol to resuspend the bacterial cells; dispense onto ice into pre-cooled EP tubes, quick-freeze in liquid nitrogen, and finally store at -80℃ to prepare Dh5a competent cells.

[0094] 1-6-2 The pBBR-sgpK plasmid was electroporated into Dh5a competent cells to prepare Dh5α / pBBR-sgpK cells:

[0095] Add 10 μL of the pBBR-sgpK plasmid to be transformed into DH5a competent cells that have been incubated on ice for 10 min. Mix well and incubate on ice for 10 min. Transfer to an electroporation dish. After electroporation, quickly remove the dish and add 1 mL of LB liquid medium. Incubate at 200 rpm and 28°C with shaking for 2 h. Spread the culture onto a kanamycin-containing plate. After 48 h of culture, collect a single colony of DH5a containing the pBBR-sgpK plasmid for culture preservation.

[0096] 1-7. Construction of the 9-OH-PCA-producing strain P. chlororaphis P3ΔH-sgpK:

[0097] 1-7-1. Preparation of P. chlororaphis P3ΔH competent cells:

[0098] (1) Pick a single colony of P. chlororaphis P3ΔH and put it into 50 mL of LB liquid medium. Incubate at 28 °C with shaking for 12 h to prepare P. chlororaphis P3ΔH bacterial suspension.

[0099] (2) Transfer the P. chlororaphis P3ΔH bacterial culture to a 50 mL centrifuge tube, let it stand on ice for 10 min, then centrifuge at 8000 rpm for 10 min, discard the supernatant, add 10% sterile glycerol to resuspend the bacterial cells, let it stand on ice for 10 min, centrifuge at 8000 rpm for 10 min, discard the supernatant, and repeat the operation once.

[0100] (4) Add 1 mL of 10% glycerol to resuspend the bacterial cells, aliquot 100 μL into 1.5 mL centrifuge tubes, store at -80℃ for later use, and prepare P. chlororaphis P3ΔH competent cells.

[0101] 1-7-2. Plasmid pBBR-sgpK was introduced into P. chlororaphis P3ΔH competent cells by electroporation:

[0102] Add 10 μL of pBBR-sgpK plasmid to be transformed into P. chlororaphis P3ΔH competent cells that have been incubated on ice for 10 min. Mix well and incubate on ice for 10 min. Transfer to an electroporation dish. After electroporation, quickly remove the dish and add 1 mL of LB liquid medium. Incubate at 200 rpm and 28 °C with shaking for 2 h. Spread the culture on a kanamycin plate and incubate for 48 h. Take a single colony for fermentation assay to obtain the P. chlororaphis P3ΔH-sgpK (P3ΔH-sgpK) strain, which is then stored for later use.

[0103] Example 2: High-yield modification of engineered strains of Pseudomonas aeruginosa to synthesize PCA derivatives.

[0104] Structural analysis and determination of mutation sites of 2-1Sgpk enzyme

[0105] The three-dimensional structure of SgpK was predicted using AlphaFold v2.0 Colab (AF, https: / / research.google.com / colaboratory). The accuracy of the AF prediction was primarily assessed using the Local Distance Difference Test (IDDT). Discovery Studio 2019 was used for docking, interaction analysis, and virtual mutation screening of the SgpK enzyme and PCA molecules. The docking structure was visualized using Pymol. The optimal SgpK enzyme variant was determined by a single-point mutation of LEU155 to LYS155 (e.g., ...). Figure 4 Structural analysis revealed enhanced hydrogen bonding at L155K and the formation of a new salt bridge with the PCA carboxyl group, thereby increasing the affinity of the SgpK enzyme for small PCA molecules (e.g., Figure 5 ).

[0106] 2-2 Construction of Sgpk enzyme mutants

[0107] The plasmid pBBR-sgpKL155K, a site-directed mutant of the sgpK gene, was constructed according to the method in Example 1. The genetically engineered strain P. chlororaphis P3ΔH-L155K (P3ΔHL155K) was constructed according to the method in Example 2. After fermentation, the yield of 9-OH-PCA was measured, and the yield exceeded that of strain P3ΔH-sgpK by 17.5%. Figure 6 The specific steps are as follows:

[0108] 2-2-1 Amplification of the sgpK-L155K site-directed mutant gene

[0109] (1) The PrimeSTAR Max DNA Polymerase kit (Shanghai Haojia Technology Development Co., Ltd.) was used. The corresponding primers were synthesized by Shanghai Ruimian Biotechnology Co., Ltd. The PCR reaction system is shown in Table 2-1.

[0110] Table 2-1 PCR amplification system (30 μL)

[0111]

[0112] (2) Double enzyme digestion of pBBR-sgpK plasmid

[0113] Using the QuickCut Dpn endonuclease kit (Shanghai Haojia Technology Development Co., Ltd.), the pBBR-sgpK plasmid vector was digested at 37℃ for 2 hours according to the double digestion system in Table 2-2.

[0114] Table 2-2 Plasmid double enzyme digestion system (50 μL)

[0115] QuickCut Dpn I 2.5 QuickCut Dpn II 2.5 10×Buffer 5 pBBR-sgpK plasmid 40

[0116] (3) Gel electrophoresis and gel recovery of PCR products and double enzyme digestion products

[0117] Using the HiPure Gel Pure DNA Mini Kit (Shanghai Chengyan Biotechnology Co., Ltd.), the PCR product from step (1) and the double-digested plasmid from step (2) were subjected to gel electrophoresis. Under blue light, the gel containing the target fragment sgpk-L155K gene fragment and the digested PBBR-sgpK plasmid fragment was excised and placed in a centrifuge tube. Solution B sol was added and the mixture was incubated at 50°C until completely dissolved. The solution was transferred to an adsorption column, centrifuged, and the filtrate was discarded. Wash twice with Wash Solution (containing alcohol), centrifuged, and the residual alcohol was dried with a hair dryer.

[0118] Add 30 μL of preheated ddH2O to the center of the membrane, centrifuge to obtain the recovered DNA solution, and store it in a -20°C freezer for subsequent ligation steps.

[0119] (4) The pBBR-sgpK plasmid was prepared by ligating the pBBR-sgpK plasmid with the site-directed mutant sgpK-L155K gene.

[0120] Using the Seamless Cloning and Assembly Kit (Beijing Adley Biotechnology Co., Ltd.), the recovered linearized vector (recovered pBBR-sgpK plasmid after enzyme digestion) was ligated with the sgpKL155 site-directed mutant gene fragment at 50℃ for 40 min according to the ligation system in Table 2-3.

[0121] Table 2-3 Connection system (10 μL)

[0122]

[0123] (5) The pBBR-sgpKL155K plasmid was introduced into P. chlororaphis P3ΔH competent cells to complete the construction.

[0124] Preparation of P. chlororaphis P3ΔH competent cells:

[0125] a. Pick a single colony of P. chlororaphis P3ΔH (Pseudomonas chlororaphis P3ΔH) and add it to 50 mL of LB liquid medium.

[0126] The P. chlororaphis P3ΔH bacterial culture was prepared by shaking culture at 28℃ for 12 h.

[0127] b. Transfer the P. chlororaphis P3ΔH bacterial culture to a 50 mL centrifuge tube, incubate on ice for 10 min, then centrifuge at 8000 rpm.

[0128] Centrifuge for 10 minutes, discard the supernatant, resuspend the bacterial cells in 10% sterile glycerol, incubate on ice for 10 minutes, then centrifuge at 8000 rpm.

[0129] Centrifuge for 10 minutes, discard the supernatant, and repeat the operation once;

[0130] c. Resuspend the bacterial cells in 1 mL of 10% glycerol, aliquot 100 μL into 1.5 mL centrifuge tubes, and store at -80°C.

[0131] Prepared P. chlororaphis P3ΔH competent cells for later use.

[0132] The plasmid pBBR-sgpKL155K was introduced into *P. chlororaphis* P3ΔH competent cells via electroporation: 10 μL of the pBBR-sgpKL155K plasmid to be transformed was added to *P. chlororaphis* P3ΔH competent cells that had been incubated on ice for 10 min. After mixing, the cells were incubated on ice for 10 min and then transferred to an electroporation dish. After electroporation, the dish was quickly removed, 1 mL of LB liquid medium was added, and the cells were incubated at 200 rpm and 28°C with shaking for 2 h. The culture was then spread onto a kanamycin-containing plate and incubated for 48 h. Single colonies were collected for fermentation assays to obtain the *P. chlororaphis* P3ΔH-L155K strain, which was then stored for future use.

[0133] Example 3: Detection of phenazine compounds in *Pseudomonas aeruginosa*

[0134] 1. Fermentation culture of engineered Pseudomonas chlororaphis P3ΔH-sgpK and P. chlororaphis P3ΔH-L155K: Using KB medium (glycerol 18.92 g / L, tryptone 20 g / L, K2HPO4 0.514 g / L, MgSO4 0.732 g / L), P. chlororaphis P3ΔH-sgpK and P. chlororaphis P3ΔH-L155K were inoculated into 5 mL of KB liquid medium and cultured overnight at 28°C and 200 rpm. The next day, the seed culture was transferred to concave-corner flasks containing 60 mL of KB liquid medium (inoculation amount according to OD). 600 The yield of phenazine derivatives was measured at approximately 0.02 g / L. The samples were cultured in a shaker at 28°C and 200 rpm for 60 h. Samples were taken every 12 h to determine the yield of phenazine derivatives. Each sample was tested in triplicate.

[0135] 2. Extraction method of phenazine derivatives: Take 1 mL of the fermentation broth from step 1 into a 4 mL centrifuge tube, adjust the pH to 2-3 with 6M HCl, then add 3 times the volume of ethyl acetate for extraction, shake thoroughly to mix, and centrifuge at 8,000 rpm for 3 min; aspirate 1 mL of the organic phase containing the target compound into a new centrifuge tube, place it in a fume hood to evaporate completely; then add 1 mL of acetonitrile to fully dissolve the compound, and filter it with a disposable filter (organic) with a pore size of 0.22 μM.

[0136] 3. Detection method for phenazine derivatives: The fermentation broth treated in step 2 was analyzed by HPLC (High Performance Liquid Chromatography). The chromatographic column was a Diamonsil C18 reverse-phase column (4.6 × 250 mm, particle size 5 μm). The mobile phase was 1‰ formic acid water and chromatographic grade acetonitrile, the flow rate was 1 mL / min (column pressure not exceeding 400 bar), and the column oven temperature was set to 25℃. HPLC detection method for 9-OH-PCA: 0-4 min, 20% acetonitrile; 4-20 min, 40% acetonitrile; 20-25 min, 20% acetonitrile; UV detection wavelength set to 270 nm. Figure 3 The results are from the HPLC detection of 9-OH-PCA.

[0137] 4. Isolation, purification, and structural identification of phenazine derivatives:

[0138] 4-1 Separation and Purification: 5 L of fermentation broth from *Pseudomonas chlororaphis* P3ΔH-sgpK and P3ΔH-L155K, capable of producing the target compound, was prepared separately. The supernatant was collected by centrifugation at 10,000 rpm for 3 min. The pH of the reaction solution was then adjusted to approximately 2.0 with hydrochloric acid, followed by extraction twice with ethyl acetate. The upper organic phase was collected. The organic phase was evaporated to dryness using a rotary evaporator at 40 °C to obtain a crude extract containing the target compound. The crude extract was dissolved in 5 mL of ethanol and then passed through a Sephadex HP-20 macroporous adsorption resin column. Impurities were first washed with 10% ethanol, followed by elution of the target compound with 30% ethanol. The 30% ethanol eluent was collected in separate test tubes and analyzed by HPLC. The eluents containing the target compound were then mixed together and evaporated to dryness by rotary evaporation. The evaporated sample was dissolved in 200 mL of acetonitrile, microfiltered, and then further separated and purified by preparative high-performance liquid chromatography (Pre-HPLC). Finally, the collected liquids with a purity greater than 95% were combined and freeze-dried.

[0139] 4-2 Structural Identification: Nuclear Magnetic Resonance Spectrometer (NMR) Detection: The purified compound was dissolved in deuterated dimethyl sulfoxide (DMSO-d6) for NMR analysis. This procedure was performed by the Analytical and Testing Center of Shanghai Jiao Tong University. Figure 7 ).

[0140] Using the method of this invention, a biosynthetic pathway for phenazine derivatives was successfully constructed in Pseudomonas aeruginosa P3 and P3ΔH, and 9-OH-PCA was prepared. These two phenazine derivatives have good antibacterial activity and can inhibit a variety of bacteria and fungi. They are expected to be developed into antibiotic drugs and provide a reference for the structural modification and microbial synthesis of other novel compounds.

[0141] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A genetically engineered bacterium producing phenazine hydroxylated derivatives, characterized in that, In Pseudomonas aeruginosa ( Pseudomonas chlororaphis Based on the heterologous expression of the sgpK gene, the genetically engineered bacteria were prepared, and the nucleotide sequence of the sgpK gene is shown in SEQ ID NO.5; the Pseudomonas aeruginosa is a PCA-producing Pseudomonas aeruginosa; the phenazine hydroxylated derivative is 9-OH-PCA.

2. The genetically engineered bacterium according to claim 1, characterized in that, The method for constructing the genetically engineered bacteria includes the following steps: S1. Extract the sgpK gene from Streptomyces griseoluteus P510; S2. The pBBR1MCS2 plasmid was double-digested with QuickCut Dpn I and QuickCut Dpn II. S3. The double-digested pBBR1MCS2 plasmid is ligated with the sgpK gene fragment to obtain the pBBR-sgpK recombinant plasmid. S4. Transform the pBBR-sgpK recombinant plasmid into Pseudomonas aeruginosa to obtain the genetically engineered bacteria.

3. The genetically engineered bacteria according to claim 2, characterized in that, In step S1, the primers for extracting the sgpK gene include sgpK-F and sgpK-R, wherein the nucleotide sequence of sgpK-F is shown in SEQ ID NO.1; and the nucleotide sequence of sgpK-R is shown in SEQ ID NO.

2.

4. A recombinant plasmid, characterized in that, The recombinant plasmid contains a site-directed mutant sgpK-L155K gene, the nucleotide sequence of which is shown in SEQ ID NO.

6.

5. A genetically engineered bacterium producing phenazine hydroxylated derivatives, characterized in that, The genetically engineered bacterium was prepared by heterologously expressing the site-directed mutant gene sgpK-L155K of the sgpK gene in *Pseudomonas aeruginosa*. The nucleotide sequence of the sgpK-L155K gene is shown in SEQ ID NO.

6. The *Pseudomonas aeruginosa* is a PCA-producing *Pseudomonas aeruginosa*. The phenazine hydroxylated derivative is 9-OH-PCA.

6. A method for constructing a genetically engineered bacterium producing phenazine hydroxylated derivatives as described in claim 5, characterized in that, Includes the following steps: Step 1: Extract the sgpK gene from Streptomyces griseoluteus P510; Step 2: Single-point mutation of LEU155 in the SgpK gene to LYS155 to obtain the SgpK-LYS155 fragment, and amplify the SgpK-LYS155 fragment. Step 3: Double digestion of the pBBR-sgpK plasmid described in claim 2 using QuickCut Dpn I and QuickCut Dpn II; Step 4: Ligate the double-digested pBBR-sgpK plasmid with the SgpK-LYS155 fragment to obtain the pBBR-sgpKL155K recombinant plasmid. Step 5: Transform the pBBR-sgpKL155K recombinant plasmid into Pseudomonas aeruginosa to obtain the genetically engineered bacteria.

7. The method for constructing a genetically engineered bacterium producing phenazine hydroxylated derivatives according to claim 6, characterized in that, In step 2, the primers used to amplify the SgpK-LYS155 fragment include L155K-F and L155K-R; the nucleotide sequence of L155K-F is shown in SEQ ID NO.3; and the nucleotide sequence of L155K-R is shown in SEQ ID NO.4.

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