Pseudomonas aeruginosa engineering bacteria with high yield of rhamnolipid, and construction method and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, and provides a high-yield rhamnolipid-producing *Pseudomonas aeruginosa* engineered bacterium, its construction method, and its applications. Compared to *Pseudomonas aeruginosa*, the key gene for polyhydroxy fatty acid (PHA) synthesis and the competing gene for extracellular polysaccharide synthesis in this engineered *P. aeruginosa* bacterium are knocked out; the key gene for PHA synthesis is... phaC1DC2 Genes; the extracellular polysaccharide synthesis competing genes include algD , pelF or pslAB Genes. This invention uses *Pseudomonas aeruginosa* PAO1 as the substrate bacterium, and after knocking out the key gene for polyhydroxy fatty acid (PHA) synthesis, it further knocks out... algD , pelF or pslAB By competing for metabolic genes, *Pseudomonas aeruginosa* engineered strains with significantly increased rhamnolipid production compared to the starting strain were screened. The *Pseudomonas aeruginosa* engineered strains described in this invention exhibit stable growth, excellent fermentation performance, and promising prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a high-yield rhamnolipid-producing Pseudomonas aeruginosa engineered bacterium, its construction method, and its application. Background Technology

[0002] Rhamnolipids are a class of glycolipid biosurfactants synthesized by microorganisms. They possess advantages such as high surface activity, complete biodegradability, good environmental compatibility, and wide availability of raw materials, making them valuable for applications in petroleum displacement, soil remediation, food processing, daily chemicals, and pharmaceuticals. *Pseudomonas aeruginosa*, with its well-developed metabolic pathways and broad substrate spectrum, is currently the dominant host for rhamnolipid synthesis.

[0003] Existing research mainly focuses on knocking out single or combined polysaccharide genes to enhance rhamnolipid synthesis. However, these studies generally suffer from problems such as unclear modification targets and insignificant yield increases. Therefore, there is an urgent need to provide a Pseudomonas aeruginosa engineered strain with clearly defined modification targets that can stably and significantly increase the yield of rhamnolipid synthesis. Summary of the Invention

[0004] The purpose of this invention is to provide a Pseudomonas aeruginosa engineered bacterium that produces high levels of rhamnolipids, its construction method, and its application. The Pseudomonas aeruginosa engineered bacterium described in this invention can significantly increase the yield of rhamnolipids and has good prospects for industrial application.

[0005] This invention provides a Pseudomonas aeruginosa engineered bacterium that produces high levels of rhamnolipids. Compared to Pseudomonas aeruginosa, the key gene for polyhydroxy fatty acid (PHA) synthesis and the competing gene for extracellular polysaccharide synthesis in the engineered bacterium have been knocked out. The key gene for the synthesis of polyhydroxy fatty acid (PHA) is... phaC1DC2 Gene; The extracellular polysaccharide synthesis competing genes include algD Gene, pelF Gene or pslAB Gene; The phaC1DC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The algD The nucleotide sequence of the gene is shown in SEQ ID NO:2; The pelF The nucleotide sequence of the gene is shown in SEQ ID NO:3; The pslAB The nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0006] As a preferred embodiment, the *Pseudomonas aeruginosa* includes *Pseudomonas aeruginosa* PAO1.

[0007] This invention also provides a method for constructing the engineered Pseudomonas aeruginosa, comprising the following steps: The knockout vector was introduced into Pseudomonas aeruginosa to obtain the engineered Pseudomonas aeruginosa strain that produces high rhamnolipids. The knockout vectors include vectors that knock out key genes for the synthesis of polyhydroxy fatty acids (PHA) and vectors that knock out competing genes for the synthesis of extracellular polysaccharides.

[0008] As a preferred embodiment, the vector for knocking out the key gene for polyhydroxy fatty acid (PHA) synthesis includes a base vector and a vector inserted into the base vector. phaC1DC2 Gene targeting fragments; The vector for knocking out the extracellular polysaccharide synthesis competing gene includes a base vector and an extracellular polysaccharide synthesis competing gene targeting fragment inserted into the base vector.

[0009] As a preferred embodiment, the phaC1DC2 The nucleotide sequence of the gene targeting fragment is shown in SEQ ID NO:23; The extracellular polysaccharide synthesis competitive gene targeting fragment includes algD Gene targeting fragments, pelF Gene targeting fragments or pslAB Gene targeting fragments; The algD The nucleotide sequence of the gene targeting fragment is shown in SEQ ID NO:24; The pelF The nucleotide sequence of the gene targeting fragment is shown in SEQ ID NO:25; The pslAB The nucleotide sequence of the gene-targeting fragment is shown in SEQ ID NO:26.

[0010] As a preferred embodiment, the base vector includes the pEX18Gm vector.

[0011] The present invention also provides the application of the engineered Pseudomonas aeruginosa or the engineered Pseudomonas aeruginosa obtained by the construction method in the preparation of rhamnolipids.

[0012] This invention also provides a fermentation method for high-yield rhamnolipid production, comprising the following steps: The engineered Pseudomonas aeruginosa was inoculated into LB liquid medium for seed culture to obtain seed liquid. The seed liquid was then inoculated into fermentation medium for fermentation culture. The obtained fermentation culture contained rhamnolipid. The fermentation medium comprises the following components: 1.0~2.0 g / L NaNO3, 0.03~0.07 g / L MgSO4·7H2O, 0.05~0.15 g / L KCl, 0.05~0.15 M sodium phosphate buffer, and 5%~15% (v / v) oil.

[0013] As a preferred method, during the fermentation process, trace elements are added to the fermentation medium every 22-26 hours; the trace elements are added at a ratio of 0.2% to 0.6% of the fermentation medium volume. The trace elements include the following components: 1.5~2.5 g / L sodium citrate, 0.25~0.3 g / L FeCl3·6H2O, 1.0~2.0 g / L ZnSO4·7H2O, 1.0~1.5 g / L CoCl2·6H2O, 1.0~1.5 g / L CuSO4·5H2O, and 0.5~1.0 g / L MnSO4·H2O.

[0014] As a preferred method, the fermentation temperature is 35~39℃, the fermentation speed is 200~240rpm, and the fermentation time is 100~140h.

[0015] Beneficial effects: This invention provides a high-yield rhamnolipid-producing *Pseudomonas aeruginosa* engineered bacterium. Compared to *Pseudomonas aeruginosa*, the key gene for polyhydroxy fatty acid (PHA) synthesis and the competing gene for extracellular polysaccharide synthesis in this engineered bacterium are knocked out. The key gene for PHA synthesis is... phaC1DC2 Genes; the extracellular polysaccharide synthesis competing genes include algD , pelF or pslAB Genes, the ones mentioned phaC1DC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; algD The nucleotide sequence of the gene is shown in SEQ ID NO:2; pelF The nucleotide sequence of the gene is shown in SEQ ID NO:3; pslAB The nucleotide sequence of the gene is shown in SEQ ID NO:4. This invention uses *Pseudomonas aeruginosa* PAO1 as the substrate bacterium, and after knocking out the key gene for polyhydroxy fatty acid (PHA) synthesis, it further knocks out… algD , pelF or pslABBy competing for metabolic genes, *Pseudomonas aeruginosa* engineered strains with significantly increased rhamnolipin production compared to the starting strain were screened. This invention clarifies the differential effects of different gene knockouts on rhamnolipin synthesis, and screens out high-yielding, stable *P. aeruginosa* engineered strains suitable for industrial production, which has significant practical implications for improving rhamnolipin fermentation levels. The screened *P. aeruginosa* engineered strains show significantly increased rhamnolipin production compared to the starting strain, and exhibit stable cell growth, suitable fermentation viscosity, high oxygen transfer efficiency, and strong genetic stability, making them directly applicable to industrial production. The modification method is simple and clear, complies with industrial microbial safety and application standards, and has good prospects for industrial application. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The images show the amplification results of the pEX18Gm-ΔphaC1DC2 plasmid construction; where A shows the amplification results of the upstream and downstream homologous arms of the PHA encoding gene (pha-up and pha-down); B shows the amplification results of the targeted fragment ΔphaC1DC2; and C shows the reverse amplification results of the pEX18Gm vector. Figure 2 Figure 1 shows the colony PCR identification results of the pEX18Gm-ΔphaC1DC2 plasmid construction and the ΔphaC1DC2 gene deletion strain construction; where A is the colony PCR identification result of the pEX18Gm-ΔphaC1DC2 plasmid constructed by homologous recombination of pEX18Gm vector and ΔphaC1DC2; and B is the colony PCR identification result of ΔphaC1DC2 after sucrose screening. Figure 3 The amplification results of the target fragments ΔalgD, ΔpelF, and ΔpslAB are shown in the figure. Figure 4 Figure 1. PCR identification results of colonies of pEX18Gm-ΔalgD, pEX18Gm-ΔpelF, and pEX18Gm-ΔpslAB plasmids constructed by homologous recombination of pEX18Gm vector with the target fragments ΔalgD, ΔpelF, and ΔpslAB. Figure 5 Figure 1 shows the colony PCR identification results of pEX18Gm-ΔphaC1DC2-ΔalgD, pEX18Gm-ΔphaC1DC2-ΔpelF, and pEX18Gm-ΔphaC1DC2-ΔpslAB after sucrose negative selection. Figure 6 The standard curve of rhamnolipids; Figure 7This is a comparison of rhamnolipin production with that of the basal strain PAO1 after fermentation culture of PAO1-ΔphaC1DC2, PAO1-ΔphaC1DC2-ΔalgD, PAO1-ΔphaC1DC2-ΔpelF, and PAO1-ΔphaC1DC2-ΔpslAB. Detailed Implementation

[0018] This invention provides a high-yield rhamnolipid-producing *Pseudomonas aeruginosa* engineered bacterium. Compared to *Pseudomonas aeruginosa*, the key gene for polyhydroxy fatty acid (PHA) synthesis and the competing gene for extracellular polysaccharide synthesis in this engineered bacterium have been knocked out. The key gene for PHA synthesis is... phaC1DC2 Genes; the extracellular polysaccharide synthesis competing genes include algD Gene, pelF Gene or pslAB Genes. Described in this invention. phaC1DC2 The genes are the core gene cluster in *Pseudomonas aeruginosa* responsible for the synthesis of polyhydroxyalkanoates (PHAs), competing with the rhamnolipid metabolism pathway for carbon sources. algD The gene is the rate-limiting gene for alginate synthesis. pelF The gene participates in the polymerization and export of the Pel polysaccharide. pslAB The gene is responsible for the initiation and transport of Psl polysaccharide. It shares a glyconucleotide precursor with rhamnolipids, resulting in strong carbon diversion and causing a large amount of carbon source to be diverted to the extracellular polysaccharide, directly limiting the synthesis efficiency and final yield of rhamnolipids. This invention uses a P. aeruginosa PHA synthesis-deficient strain as the starting strain to reduce carbon diversion and then knocks out the gene by blocking the competitive metabolic pathway. pslAB , pelF or algD Three carbon flow competing genes can efficiently increase the production of rhamnolipin in Pseudomonas aeruginosa engineered bacteria.

[0019] As one specific implementation method, the phaC1DC2

[0020] As one specific implementation method, the algD

[0021] As one specific implementation method, the pelF

[0022] As one specific implementation method, the pslAB

[0023] The *Pseudomonas aeruginosa* strain described in this invention includes *Pseudomonas aeruginosa* PAO1. As a specific embodiment, *Pseudomonas aeruginosa* PAO1 can be obtained through purchase; the *Pseudomonas aeruginosa* PAO1 described in this invention was purchased from Hangzhou Baosai Biotechnology Co., Ltd. The accession number for *Pseudomonas aeruginosa* PAO1 described in this invention is NC_002516.2.

[0024] The present invention also provides a method for constructing the engineered Pseudomonas aeruginosa, comprising the following steps: introducing a knockout vector into Pseudomonas aeruginosa to obtain the engineered Pseudomonas aeruginosa with high rhamnolipid production; the knockout vector includes a vector that knocks out a key gene for the synthesis of polyhydroxy fatty acids (PHA) and a vector that knocks out a competing gene for the synthesis of extracellular polysaccharides.

[0025] As one specific implementation, the vector for knocking out the key gene for polyhydroxy fatty acid (PHA) synthesis includes a base vector and a vector inserted into the base vector. phaC1DC2 Gene targeting fragment; the vector for knocking out extracellular polysaccharide synthesis competing genes includes a base vector and an extracellular polysaccharide synthesis competing gene targeting fragment inserted into the base vector. As a specific embodiment, the base vector includes the pEX18Gm vector. As a specific embodiment, the extracellular polysaccharide synthesis competing gene targeting fragment includes... algD Gene targeting fragments, pelF Gene targeting fragments or pslAB Gene targeting fragments. As one specific implementation, the base vector includes the pEX18Gm vector.

[0026] As one specific implementation method, the phaC1DC2

[0027] As one specific implementation method, the algD

[0028] As one specific implementation method, the pelF

[0029] As one specific implementation method, the pslAB

[0030] This invention does not specifically limit the preparation method of the target fragment; any conventional method in the art can be used. phaC1DC2 Taking gene targeting fragments as an example, in this invention, the... phaC1DC2 Methods for preparing gene targeting fragments include: separately amplifying... phaC1DC2 The upstream and downstream homologous arms of the gene are obtained. phaC1DC2 upstream homologous arm gene fragments and phaC1DC2 The downstream homologous arm gene fragment; will phaC1DC2 upstream homologous arm gene fragments and phaC1DC2 The downstream homologous arm gene fragments of the gene are ligated to obtain phaC1DC2 Gene targeting fragments.

[0031] get phaC1DC2 After gene targeting fragments, this invention will phaC1DC2 The gene targeting fragment is cloned into a basic vector. This invention does not specifically limit the cloning method; any conventional cloning method in the art can be used. In this invention, the basic vector includes the pEX18Gm vector, which carries the gentamicin resistance gene (…). GmR ) as a positive screening marker and sacB Genes are used as negative selection markers. sacB The gene encodes fructanase, which catalyzes the hydrolysis or polymerization of sucrose in the presence of sucrose to form fructans, which are toxic to cells. sacB Cells cannot grow. A two-step selection strategy, using positive selection (gentamicin resistance) and negative selection (sucrose sensitivity), is employed to obtain strains with the target gene knocked out. In this invention, the cloning method includes: […]. phaC1DC2 The gene targeting fragment and the pEX18Gm linearized vector were mixed at a molar ratio of 2:1, homologous recombinase was added, and ddH2O was added to bring the volume to 20 μL. After reacting at 50°C for 1 h, the mixture was transformed into DH5α competent cells and cultured overnight at 37°C. The vector pEX18Gm-ΔphaC1DC2, which knocks out the key gene for PHA synthesis, was obtained after identification. The pEX18Gm vector described in this invention was purchased from Shutong Technology.

[0032] After obtaining a vector that knocks out the key gene for PHA synthesis, this invention transforms the vector into competent E. coli cells, and constructs a gene that knocks out the key gene for PHA synthesis through conjugation transfer. phaC1DC2Gene-deleted strains. This invention does not specifically limit the transformation method; any conventional transformation method in the art can be used. This invention uses a chemical transformation method to transform a vector that knocks out the key gene for polyhydroxy fatty acid (PHA) synthesis into competent *E. coli* cells. The chemical transformation method includes: transforming the pEX18Gm-ΔphaC1DC2 plasmid into *E. coli* SM10-λpir chemically competent cells. The procedure is as follows: incubation on ice for 30 min, heat shock at 42°C for 45 s, addition of 1 mL of antibiotic-free liquid LB medium, and incubation at 37°C and 220 rpm for 1 h. The cells are then plated on solid LB plates containing 10 μg / mL and incubated overnight at 37°C to obtain single colonies containing the pEX18Gm-ΔphaC1DC2 plasmid.

[0033] After obtaining single colonies containing the pEX18Gm-ΔphaC1DC2 plasmid, this invention constructs a system by conjugating and transferring the key gene for the synthesis of polyhydroxy fatty acids (PHA) to knock out the PHA. phaC1DC2 Gene-deleted strains. The conjugation transfer method includes: picking single colonies of bacteria cultured overnight and single colonies of *Pseudomonas aeruginosa* PAO1 in LB liquid medium and culturing at 220 rpm and 37°C until OD. 600 The concentration of the two bacterial cultures was 0.6. They were mixed at a 1:1 ratio and inoculated onto antibiotic-free LB agar plates, then incubated at 30°C overnight. The bacterial colony was collected, resuspended, and spread onto LB agar plates containing 10 μg / mL gentamicin. The plates were incubated at 37°C for 48 h. Single-crossover strains with plasmid integration into the chromosome were screened to obtain the phaC1DC2 knockout strain PAO1-ΔphaC1DC2.

[0034] After obtaining the successfully knocked-out phaC1DC2 strain PAO1-ΔphaC1DC2, the above-described knockout method was followed on the PAO1-ΔphaC1DC2 strain. algD Genes were extracted to obtain PAO1-ΔphaC1DC2ΔalgD engineered bacteria; the bacteria were then knocked out according to the above method. pelF Genes were extracted to obtain PAO1-ΔphaC1DC2-ΔpelF engineered bacteria; the bacteria were then knocked out according to the above method. pslAB Genes were used to obtain PAO1-ΔphaC1DC2-ΔpslAB engineered bacteria.

[0035] The engineered bacteria of this invention can block the extracellular polysaccharide competitive pathway, achieve precise carbon flow redirection, and more efficiently allocate carbon sources to rhamnolipide synthesis. The rhamnolipide yield of the engineered bacteria PAO1-ΔphaC1DC2-ΔalgD with the rate-limiting gene for alginate synthesis knocked out is about 20% higher than that of the original strain PAO1-ΔphaC1DC2. The rhamnolipide yield of the engineered bacteria PAO1-ΔphaC1DC2-ΔpslAB with the gene responsible for the initiation and transport of Psl polysaccharide knocked out is about 75% higher than that of the original strain PAO1-ΔphaC1DC2.

[0036] The present invention also provides the application of the engineered Pseudomonas aeruginosa or the engineered Pseudomonas aeruginosa obtained by the construction method in the preparation of rhamnolipids.

[0037] This invention also provides a fermentation method for high-yield rhamnolipid production, comprising the following steps: The engineered Pseudomonas aeruginosa was inoculated into LB liquid medium for seed culture. After obtaining the seed liquid, the seed liquid was inoculated into fermentation medium for fermentation culture. The obtained fermentation culture contained rhamnolipid.

[0038] In this invention, the engineered Pseudomonas aeruginosa is first inoculated into LB liquid medium for seed culture to obtain seed culture. In one preferred embodiment, the inoculation volume of the seed culture is 0.5% to 15% of the culture medium volume, and the fermentation culture volume is 45 to 55 mL; in another preferred embodiment, the inoculation volume of the seed culture is 1% of the culture medium volume, and the fermentation culture volume is 50 mL.

[0039] After obtaining the seed liquid, the seed liquid is inoculated into a fermentation medium for fermentation culture, and the resulting fermentation culture contains rhamnolipin.

[0040] The fermentation medium of the present invention comprises the following components: 1.0~2.0 g / L NaNO3, 0.03~0.07 g / L MgSO4·7H2O, 0.05~0.15 g / L KCl, 0.05~0.15 M sodium phosphate buffer, and 5%~15% (v / v) oil. In a preferred embodiment, the fermentation medium comprises the following components: 1.2~1.8 g / L NaNO3, 0.04~0.06 g / L MgSO4·7H2O, 0.08~0.12 g / L KCl, 0.08~0.12 M sodium phosphate buffer, and 8%~12% (v / v) oil. In another preferred embodiment, the fermentation medium comprises the following components: 1.5 g / L NaNO3, 0.05 g / L MgSO4·7H2O, 0.1 g / L KCl, 0.1 M sodium phosphate buffer, and 10% (v / v) oil.

[0041] In the fermentation process described in this invention, trace elements are added to the fermentation medium every 22-26 hours. As a specific implementation, the interval for adding trace elements can be any value from 22h, 23h, 24h, 25h, and 26h, or the midpoint of any two of these values. The trace elements are added at a ratio of 0.2% to 0.6% of the fermentation medium volume. As a specific implementation, the amount of trace elements added can be any value from 0.2%, 0.3%, 0.4%, 0.5%, and 0.6%, or the midpoint of any two of these values. This invention adds trace elements to the fermentation medium every 22-26 hours to continuously supply essential cofactors for enzymatic reactions, ensuring the energy metabolism and fatty acid degradation efficiency of *Pseudomonas aeruginosa*, maintaining the activity of the rhamnolipid synthesis pathway, and avoiding growth restriction and metabolic disorders caused by metal ion depletion, thereby achieving stable strain growth and efficient synthesis of the target product.

[0042] The trace elements described in this invention comprise the following components: 1.5~2.5 g / L sodium citrate, 0.25~0.3 g / L FeCl3·6H2O, 1.0~2.0 g / L ZnSO4·7H2O, 1.0~1.5 g / L CoCl2·6H2O, 1.0~1.5 g / L CuSO4·5H2O, and 0.5~1.0 g / L MnSO4·H2O. In a preferred embodiment, the trace elements comprise the following components: 1.8~2.2 g / L sodium citrate, 0.26~0.29 g / L FeCl3·6H2O, 1.2~1.8 g / L ZnSO4·7H2O, 1.1~1.3 g / L CoCl2·6H2O, 1.3~1.5 g / L CuSO4·5H2O, and 0.6~0.9 g / L MnSO4·H2O. MnSO4·H2O; In a preferred embodiment, the trace elements include the following components: 2.0 g / L sodium citrate, 0.28 g / L FeCl3·6H2O, 1.4 g / L ZnSO4·7H2O, 1.2 g / L CoCl2·6H2O, 1.2 g / L CuSO4·5H2O and 0.8 g / L MnSO4·H2O.

[0043] The fermentation culture temperature of this invention is 35~39℃, the fermentation rotation speed is 200~240 rpm, and the fermentation time is 100~140 h. As a specific embodiment, the fermentation temperature can be any value from 35℃, 36℃, 37℃, 38℃, and 39℃, or any intermediate value between any two of these values. As a specific embodiment, the fermentation rotation speed can be any value from 200 rpm, 210 rpm, 220 rpm, 230 rpm, and 240 rpm, or any intermediate value between any two of these values. As a specific embodiment, the fermentation time can be any value from 100 h, 110 h, 120 h, 130 h, and 140 h, or any intermediate value between any two of these values.

[0044] The engineered strain PAO1-ΔphaC1DC2, developed in this invention, showed a 115% increase in rhamnolipin production compared to the wild-type PAO1-WT. The engineered strain PAO1-ΔphaC1DC2-ΔpelF showed a 90% increase in rhamnolipin production compared to the wild-type, but a slight decrease of approximately 17.8% compared to the original strain. This may be because knocking out pelF in *Pseudomonas aeruginosa* inhibits the synthesis of extracellular polysaccharides in *Pel*, leading to intracellular accumulation of UDP-glucose and reducing the supply of rhamnosine precursors through feedback inhibition. Simultaneously, biofilm disruption and decreased intracellular c-di-GMP levels inhibited the rhl quorum sensing system and the transcription of rhamnolipin synthesis genes, ultimately resulting in a significant reduction in rhamnolipin production. However, PAO1-ΔphaC1DC2-ΔalgD and PAO1-ΔphaC1DC2-ΔpslAB both increased rhamnolipin production compared to the original strain, by 20% and 75%, respectively, demonstrating promising prospects for industrial application.

[0045] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a high-rhamnolipid-producing Pseudomonas aeruginosa engineered bacterium, its construction method, and its applications, should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, and commercially available products well known to those skilled in the art can be used.

[0047] The gene editing method described in this embodiment of the invention includes a two-step allele exchange method mediated by the pEX18Gm suicide vector; The DNA gel recovery kit and Taq DNA polymerase used in the embodiments of this invention were purchased from Thermo Fisher Scientific, and the homologous recombinase was purchased from Ibotek Biotechnology Co., Ltd. Pseudomonas aeruginosa strain PAO1 was purchased from Hangzhou Baosai Biotechnology Co., Ltd. Liquid LB medium: Each liter of LB medium contains 5g of yeast extract, 10g of peptone, and 10g of sodium chloride; Solid LB medium: Add 20g of agar to each liter of liquid LB medium; Fermentation medium: Each liter of the fermentation medium preferably contains 1.5 g / L NaNO3, 0.05 g / L MgSO4·7H2O, 0.1 g / L KCl, 0.1 M sodium phosphate buffer (pH 6.5) and 10% waste oil.

[0048] Example 1: Construction of pEX18Gm-ΔphaC1DC2 plasmid 1. Obtain the pha-up and pha-down gene fragments of the upstream and downstream homologous arms of the polyhydroxy fatty acid (PHA) encoding gene. Using the genome of *Pseudomonas aeruginosa* strain PAO1 (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) as a template, PCR amplification was performed using the upstream homologous arm primer pair pha-up-F / pha-up-R and the downstream homologous arm primer pair pha-down-F / pha-down-R, respectively. The PCR products were identified by nucleic acid electrophoresis and purified by gel extraction to obtain the upstream homologous arm gene fragment pha-up and the downstream homologous arm gene fragment pha-down of the polyhydroxy fatty acid (PHA) encoding gene. The PCR amplification primer sequences are shown below, and the nucleic acid electrophoresis identification results are as follows. Figure 1 As shown in Figure A.

[0049] pha-up-F: 5'-CCGAAGGAAGACCATGATTACATGAGTCAGAAGAACAATAACGAG-3' (SEQ IDNO: 5); pha-up-R: 5'-GCGTGTTGTCGTTGTTCCAGTAGGTCCTTGGCCAGGTGGCCG-3' (SEQ ID NO: 6); pha-down-F: 5'-CTCGGCCACCTGGCCAAGGACCTACTGGAACAACGACAACACG-3' (SEQ IDNO: 7); pha-down-R: 5'-GTAAAACGACGGCCAGTGCCCAGCGTATATGCACGTAGGTG-3' (SEQ ID NO: 8); The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, 34 cycles; 72℃ extension for 5 min.

[0050] 2. Obtain the target shooting fragment ΔphaC1DC2 The templates were pha-up and pha-down fragments purified by gel recovery, and the primers were SEQ ID NO:5 and SEQ ID NO:8; The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 40 s, 34 cycles; 72℃ final extension for 5 min. The PCR products were identified by nucleic acid electrophoresis and purified by gel extraction to obtain the recovered product of the targeted fragment ΔphaC1DC2. The amplification results are as follows: Figure 1 As shown in B.

[0051] 3. Obtain and construct the pEX18Gm vector Using pEX18GM plasmid (purchased from Zhuhai Shutong Medical Technology Co., Ltd.) as a template, the pEX18Gm vector gene fragment was amplified by reverse PCR. The primers used for amplification were pEX18GM-VF: 5'-GGCACTGGCCGTCGTTTTACAACGTCGTGAC-3' (SEQ ID NO:9) and pEX18GM-VR: 5'-GTAATCATGGTCTTCCTTCGGCTGGCGCTGCG-3' (SEQ ID NO:10).

[0052] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 4 min, 34 cycles; and a final extension at 72℃ for 5 min. The PCR products were identified by nucleic acid electrophoresis and purified by gel extraction to obtain the linearized pEX18GM plasmid. The amplification results are as follows: Figure 1 As shown in C.

[0053] 4. Construct the pEX18Gm-ΔphaC1DC2 plasmid via homologous recombination. The obtained targeting fragment ΔphaC1DC2 was mixed with the pEX18Gm linearized vector at a molar ratio of 2:1. Homologous recombinase was added, and ddH2O was added to bring the volume to 20 μL. After incubation at 50°C for 1 h, the mixture was transformed into DH5α competent cells and cultured overnight at 37°C. Single clones were picked for colony PCR using primers SEQ ID NO:5 and SEQ ID NO:8. The colony PCR identification results are as follows: Figure 2 As shown in Figure A. The colony PCR amplification program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min 30 s, 34 cycles; 72℃ further extension for 5 min, resulting in a 1000 bp colony PCR band. Samples with correct bands are sent for sequencing; correct sequencing indicates successful construction, yielding the pEX18Gm-ΔphaC1DC2 plasmid.

[0054] Example 2: phaC1DC2 Construction of gene deletion strains Allele exchange was performed using the suicide vector pEX18Gm to knock out the PHA synthesis gene, and a new gene was constructed. phaC1DC2 Gene deletion strain 1. Transform the pEX18Gm-ΔphaC1DC2 plasmid into E. coli SM10-λpir competent cells. The pEX18Gm-ΔphaC1DC2 plasmid was transformed into Escherichia coli SM10-λpir chemocompetent cells via chemical transformation. The procedure was as follows: incubation on ice for 30 min, heat shock at 42°C for 45 s, addition of 1 mL of antibiotic-free liquid LB medium, and incubation at 37°C and 220 rpm for 1 h. The cells were then plated on solid LB plates containing 10 μg / mL and incubated at 37°C overnight.

[0055] 2. Joining transfer Pick a single colony from the overnight culture in step 1 (as the donor bacterium), and simultaneously pick a single colony of *Pseudomonas aeruginosa* PAO1 (as the recipient bacterium) in LB liquid medium, and incubate at 220 rpm and 37°C until OD500. 600 The concentration was 0.6. The two bacterial cultures were mixed at a 1:1 ratio and inoculated onto antibiotic-free LB agar plates. Incubate at 30℃ overnight. Collect bacterial colonies, resuspend them, and spread them on LB agar plates containing 10 μg / mL gentamicin. Incubate at 37℃ for 48 h to screen for single-crossover strains with plasmid integration into the chromosome.

[0056] 3. Screening of double-exchange strains using sucrose plates Single colonies were picked and inoculated onto antibiotic-free LB liquid medium and incubated overnight at 37°C with shaking. The bacterial suspension was diluted and spread onto salt-free LB agar plates containing 15% (w:v) sucrose. The plates were incubated at 28°C for 3 days. Single colonies growing on the sucrose plates were then spotted onto LB agar plates containing 30 μg / mL gentamicin and antibiotic-free LB agar plates, respectively, to screen for gentamicin-sensitive clones.

[0057] Colonies were picked from gentamicin plates and colony PCR was performed using primer pairs SEQ ID NO:5 and SEQ ID NO:8. The colony PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min 30 s, 34 cycles; 72℃ final extension for 5 min. The successfully knocked-out amplified fragment was approximately 1000 bp in size. Figure 2 As shown in B.

[0058] Samples with the correct band were selected for sequencing. If the sequencing was correct, the knockout was successful, yielding the PAO1-ΔphaC1DC2 strain. This strain was then used as the starting strain, i.e., the recipient strain for conjugation transfer, for further processing. algD , pelF or pslAB Gene knockout.

[0059] Example 3: Construction of pEX18Gm-ΔalgD, pEX18Gm-ΔpelF and pEX18Gm-ΔpslAB plasmids 1. Obtained separately algD , pelF , pslAB The target shooting segments ΔalgD, ΔpelF, ΔpslAB Two rounds of PCR amplification were performed: the first round amplified the... algD , pelF , pslAB The upstream and downstream homologous arms of the gene, algD-up, algD-down, pelF-up, pelF-down, and pslAB-up and pslAB-down, were amplified using their respective purified up / down arms as templates and up-F and down-R primers to amplify the target fragments ΔalgD, ΔpelF, and ΔpslAB. The specific steps are as follows: algD The primer pairs for amplifying the upstream and downstream homologous arms of the gene are as follows: algD-up-F (SEQ ID NO:11): 5'-CCGAAGGAAGACCATGATTACATGCGAATCAGCATCTTTGGTTTG-3'; algD-up-R (SEQ ID NO: 12): 5'-GATAGGTGAGGGCGCGTACAGATCGCGGTGCTCTCGCGGAG-3'; algD-down-F (SEQ ID NO:13): 5'-CCTCCGCGAGAGCACCGCGATCTGTACGCGCCCTCACCTATC-3'; algD-down-R (SEQ ID NO:14): 5'-GTAAAACGACGGCCAGTGCCTACCAGCAGATGCCCTCGGCCTG-3'; pelF The primer pairs for amplifying the upstream and downstream homologous arms of the gene are as follows: pelF-up-F (SEQ ID NO:15): 5'-CCGAAGGAAGACCATGATTACTCATGCAATCTCCGTGGCTTCG-3'; pelF-up-R (SEQ ID NO:16): 5'-CTTCTGGACCCTGCGCTCGAAGCGCGATGCCGGAGGCGG-3'; pelF-down-F (SEQ ID NO:17): 5'-CCGCCTCCGGCATCGCGCTTCGAGCGCAGGGTCCAGAAG-3'; pelF-down-R (SEQ ID NO:18): 5'-GTAAACGACGGCCAGTGCCTGACCGAACACACCGCTCCGACGG-3'; pslAB The primer pairs for amplifying the upstream and downstream homologous arms of the gene are as follows: pslAB-up-F (SEQ ID NO:19): 5'-CGAAGGAAGACCATGATTACTCTTCAAGTTCCGCTCGATGTACAC-3'; pslAB-up-R (SEQ ID NO:20): 5'-GGGGAAGTCGCCGACGGCCGAGGCGGGTCCCGGAAC-3'; pslAB-down-F (SEQ ID NO:21): 5'-GTTCCGGGACCCGCCTCGGCCGTCGGCGACTTCCCC-3'; pslAB-down-R (SEQ ID NO:22): 5'-GTAAAACGACGGCCAGTGCCGCGGGGGTGTTGCATGCGGCCAG-3'.

[0060] The PCR system used for amplifying the ΔalgD target fragment was as follows: 1 µl primer SEQ ID NO:11, 1 µl primer SEQ ID NO:14, 25 µl PrimeSTAR® Max DNA Polymerase, 100 ng template, and water to a final volume of 50 µl. The PCR amplification program was as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing for 15 s, 72°C extension for 40 s, 34 cycles; and a final extension at 72°C for 5 min.

[0061] The system and procedure for amplifying the target fragment ΔpelF are the same as those for the target fragment ΔalgD, except that the primers are replaced with SEQ ID NO:15 and SEQ ID NO:18.

[0062] The system and procedure for amplifying the target fragment ΔpslAB are the same as those for the target fragment ΔalgD, except that the primers are replaced with SEQ ID NO:19 and SEQ ID NO:22.

[0063] After amplification, the purified fragment was obtained using a gel extraction kit. The amplification results are as follows: Figure 3 As shown.

[0064] The plasmids pEX18Gm-ΔalgD, pEX18Gm-ΔpelF, and pEX18Gm-ΔpslAB were constructed using the same method as the pEX18Gm-ΔphaC1DC2 plasmid in Example 1. The primers used for pEX18Gm-ΔalgD colony PCR were SEQ ID NO:11 and SEQ ID NO:14; the primers used for pEX18Gm-ΔpelF colony PCR were SEQ ID NO:15 and SEQ ID NO:18; and the primers used for pEX18Gm-ΔpslAB colony PCR were SEQ ID NO:19 and SEQ ID NO:22. The identification results are as follows: Figure 4 As shown.

[0065] Example 4: algD Gene deletion strains, pelF Gene deletion strains and pslAB Construction of gene deletion strains Allelic exchange was performed using the suicide vector pEX18Gm, with PAO1-ΔphaC1DC2 as the starting strain, to construct... algD , pelF or pslAB Gene deletion strain The method was consistent with the construction of the PAO1-ΔphaC1DC2 strain. The primers used for PAO1-ΔphaC1DC2-ΔalgD colony PCR were SEQ ID NO:11 and SEQ ID NO:14; the primers used for PAO1-ΔphaC1DC2-ΔpelF colony PCR were SEQ ID NO:15 and SEQ ID NO:18; and the primers used for PAO1-ΔphaC1DC2-ΔpslAB colony PCR were SEQ ID NO:19 and SEQ ID NO:22. The colony PCR identification results are as follows: Figure 5As shown. Select the correct samples for sequencing. After successful sequencing, the following strains were obtained: PAO1-ΔphaC1DC2-ΔalgD, PAO1-ΔphaC1DC2-ΔpelF, and PAO1-ΔphaC1DC2-ΔpslAB.

[0066] Example 5: Obtaining rhamnolipids by fermentation culture of engineered bacteria 1. Seed culture Five engineered bacteria, namely PAO1-WT, PAO1-ΔphaC1DC2 obtained in Example 2, PAO1-ΔphaC1DC2-ΔalgD strain obtained in Example 4, PAO1-ΔphaC1DC2-ΔpelF strain, and PAO1-ΔphaC1DC2-ΔpslAB strain, were streaked on antibiotic-free LB agar plates. Single colonies were picked and added to 20 mL of liquid LB medium and cultured overnight at 37°C and 220 rpm to obtain the engineered bacterial seed culture.

[0067] 2. Fermentation culture The seed cultures of the five engineered bacteria described in step 1 were added to 50 mL of fermentation medium, which consisted of 1.5 g / L NaNO3, 0.05 g / L MgSO4·7H2O, 0.1 g / L KCl, 0.1 M sodium phosphate buffer (pH 6.5), and 10% waste oil. The inoculum size was 1%, and the fermentation time was 120 h. Samples were taken every 4 h to determine the OD of the bacterial cells. 600 The growth curve was measured. 0.4% trace elements were added every 24 hours. The trace element formula was 2.0 g / L sodium citrate·2H2O, 0.28 g / L FeCl3·6H2O, 1.4 g / L ZnSO4·7H2O, 1.2 g / L CoCl2·6H2O, 1.2 g / L CuSO4·5H2O, and 0.8 g / L MnSO4·H2O. After the ultrapure water was prepared, it was filtered through a 0.22 μm aqueous filter membrane.

[0068] Example 6: Monitoring of rhamnolipid content 1. Detection Principle The sulfuric acid-anthrone method is a classic colorimetric method for determining the glycosyl content in glycolipid biosurfactants (such as rhamnolipids). The core principle is to utilize the colorimetric reaction between the dehydration products of sugars and anthrone under strong acid conditions, allowing for indirect determination of rhamnolipid concentration through colorimetric quantification. Under heating conditions, concentrated sulfuric acid causes the rhamnosyl groups in the rhamnolipid molecule to undergo a dehydration reaction, generating hydroxymethylfurfural (5-hydroxymethyl-2-furfural). Hydroxymethylfurfural condenses with anthrone reagent (anthrone is soluble in concentrated sulfuric acid) to form a blue-green anthrone sugar derivative. This substance has a characteristic absorption peak at a wavelength of 620 nm, and its absorbance is directly proportional to the sugar content.

[0069] 2. Preparation of sulfuric acid-anthrone reagent Weigh 0.2g of anthrone and dissolve it in 100mL of 98% concentrated sulfuric acid. After it is fully dissolved, let it stand in the dark until it becomes clear.

[0070] 3. Determination of standard curve 1) Take 1 g / L rhamnose standard solution and dilute it with ultrapure water to prepare a solution with the following concentration: Take 1.0 mL of each of the standard working solutions with concentrations of 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, and 0.6 mg / mL and add them to a 10 mL stoppered colorimetric tube.

[0071] 2) Slowly add 4.0 mL of anthrone reagent to each colorimetric tube, stopper the tube, and gently invert to mix.

[0072] 3) Place the colorimetric tube in a boiling water bath and heat for 10 minutes. Remove it and immediately cool it to room temperature in an ice bath. Let it stand for 10 minutes.

[0073] 4) Using a 0 mg / L standard tube as a blank control, the absorbance of each tube was measured at a wavelength of 620 nm.

[0074] 5) A standard curve was plotted with rhamnose concentration on the x-axis and absorbance on the y-axis, yielding the linear regression equation Y = 1.336X + 0.004264. (See...) Figure 6 .

[0075] 4. Determination of rhamnolipid content The five fermentation broths from Example 5 were centrifuged at 8000 rpm for 10 min, and the supernatant was collected.

[0076] 1) Take 100 μL of sample, add 1 mL of ethyl acetate and mix thoroughly for extraction. After extraction, centrifuge at 12000 rpm for 5 min, remove 500 μL of the upper ethyl acetate phase and dry. 2) Redissolve the dried sample in 1 mL of pure water, vortex thoroughly, and dilute as needed. The final sample volume is 1 mL. 3) Add 4 mL of anthrone reagent, react at 100 °C for 10 min, and then place in an ice bath for 10 min after the reaction is complete; 4) OD 620nm Measure the absorbance, subtract the blank value, and substitute it into the standard curve to calculate the concentration.

[0077] The results are as follows Figure 7As shown in the results, the engineered strain PAO1-ΔphaC1DC2, fermented in this invention, produced rhamnolipin at a yield approximately 115% higher than the wild-type PAO1-WT, and the engineered strain PAO1-ΔphaC1DC2-ΔpelF produced rhamnolipin at a yield 90% higher than the wild-type. However, there was a slight decrease compared to the original strain, approximately 17.8%. This may be because knocking out pelF in *Pseudomonas aeruginosa* inhibited the synthesis of extracellular polysaccharides in *Pel*, leading to the accumulation of UDP-glucose intracellularly. This accumulation reduced the supply of rhamnolipin precursors through feedback inhibition. Simultaneously, the disruption of the biofilm structure and the decrease in intracellular c-di-GMP levels inhibited the rhl quorum sensing system and the transcription of rhamnolipin synthesis genes, ultimately resulting in a significant reduction in rhamnolipin production. PAO1-ΔphaC1DC2-ΔalgD and PAO1-ΔphaC1DC2-ΔpslAB both increased rhamnolipin production compared to the original strain, by 20% and 75%, respectively, demonstrating promising prospects for industrial application.

[0078] Therefore, it can be seen that this invention uses Pseudomonas aeruginosa PAO1 as the substrate bacteria, and after knocking out the key gene cluster PAO1-ΔphaC1DC2 for the synthesis of polyhydroxy fatty acids (PHA), it knocks out... algD , pelF or pslAB Three single-gene deletion engineered strains were obtained by identifying three key competing metabolic genes. This invention elucidates the differential regulatory effects of gene knockout on rhamnolipid synthesis in different exopolysaccharide synthesis pathways, and screens for engineered strains with significantly increased rhamnolipid production compared to the starting strains. The obtained strains exhibit stable growth, excellent fermentation performance, and promising prospects for industrial application.

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A high-yield rhamnolipid-producing Pseudomonas aeruginosa engineered bacterium, characterized in that, Compared to Pseudomonas aeruginosa, the key gene for polyhydroxy fatty acid (PHA) synthesis and the competing gene for extracellular polysaccharide synthesis in the engineered Pseudomonas aeruginosa strain were knocked out. The key gene for the synthesis of polyhydroxy fatty acid (PHA) is... phaC1DC2 Gene; The extracellular polysaccharide synthesis competing genes include algD Gene, pelF Gene or pslAB Gene; The phaC1DC2 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The algD The nucleotide sequence of the gene is shown in SEQ ID NO:2; The pelF The nucleotide sequence of the gene is shown in SEQ ID NO:3; The pslAB The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

2. The engineered Pseudomonas aeruginosa according to claim 1, characterized in that, The *Pseudomonas aeruginosa* mentioned includes *Pseudomonas aeruginosa* PAO1.

3. The method for constructing the engineered Pseudomonas aeruginosa as described in claim 1 or 2, characterized in that, Includes the following steps: The knockout vector was introduced into Pseudomonas aeruginosa to obtain the engineered Pseudomonas aeruginosa strain that produces high rhamnolipids. The knockout vectors include vectors that knock out key genes for the synthesis of polyhydroxy fatty acids (PHA) and vectors that knock out competing genes for the synthesis of extracellular polysaccharides.

4. The construction method according to claim 3, characterized in that, The vector for knocking out the key gene in the synthesis of polyhydroxy fatty acid (PHA) includes a base vector and an insertion point into the base vector. phaC1DC2 Gene targeting fragments; The vector for knocking out the extracellular polysaccharide synthesis competing gene includes a base vector and an extracellular polysaccharide synthesis competing gene targeting fragment inserted into the base vector.

5. The construction method according to claim 4, characterized in that, The phaC1DC2 The nucleotide sequence of the gene targeting fragment is shown in SEQ ID NO:23; The extracellular polysaccharide synthesis competitive gene targeting fragment includes algD Gene targeting fragments, pelF Gene targeting fragments or pslAB Gene targeting fragments; The algD The nucleotide sequence of the gene targeting fragment is shown in SEQ ID NO:24; The pelF The nucleotide sequence of the gene targeting fragment is shown in SEQ ID NO:25; The pslAB The nucleotide sequence of the gene-targeting fragment is shown in SEQ ID NO:

26.

6. The construction method according to claim 4 or 5, characterized in that, The underlying vector includes the pEX18Gm vector.

7. The use of the engineered Pseudomonas aeruginosa according to claim 1 or 2, or the engineered Pseudomonas aeruginosa obtained by the construction method according to any one of claims 3 to 6, in the preparation of rhamnolipids.

8. A fermentation method for high-yield rhamnolipid production, characterized in that, Includes the following steps: The Pseudomonas aeruginosa engineered bacteria of claim 1 is inoculated into LB liquid medium for seed culture. After obtaining the seed liquid, the seed liquid is inoculated into fermentation medium for fermentation culture. The obtained fermentation culture contains rhamnolipid. The fermentation medium comprises the following components: 1.0~2.0 g / L NaNO3, 0.03~0.07 g / L MgSO4·7H2O, 0.05~0.15 g / L KCl, 0.05~0.15 M sodium phosphate buffer, and 5%~15% (v / v) oil.

9. The fermentation method according to claim 8, characterized in that, During the fermentation process, trace elements are added to the fermentation medium every 22-26 hours; the trace elements are added at a ratio of 0.2% to 0.6% of the fermentation medium volume. The trace elements include the following components: 1.5~2.5 g / L sodium citrate, 0.25~0.3 g / L FeCl3·6H2O, 1.0~2.0 g / L ZnSO4·7H2O, 1.0~1.5 g / L CoCl2·6H2O, 1.0~1.5 g / L CuSO4·5H2O, and 0.5~1.0 g / L MnSO4·H2O.

10. The fermentation method according to claim 8, characterized in that, The fermentation temperature is 35~39℃, the fermentation speed is 200~240rpm, and the fermentation time is 100~140h.

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