Recombinant pseudomonas engineering bacteria for producing rhamnolipid, construction method and application thereof

Abstract

The application discloses a recombinant Pseudomonas engineering bacterium for producing rhamnolipid, a construction method and application thereof. The recombinant Pseudomonas engineering bacterium is constructed by introducing a recombinant plasmid into a Pseudomonas host bacterium; the recombinant plasmid contains an arabinose inducible promoter and a lipase gene regulated by the arabinose inducible promoter lipA and a molecular chaperone lipH ; the lipA is lipH co-expressed in the host bacterium; the host bacterium is Pseudomonas aeruginosa or a Pseudomonas putida engineering bacterium integrated with a rhamnolipid synthesis pathway. The application uses a self-constructed Pseudomonas engineering bacterium as a chassis cell to construct a recombinant bacterium. In fermentation, the expression is induced by a specific concentration of arabinose, thereby improving the rapeseed oil utilization capacity of the Pseudomonas aeruginosa. In particular, the safe chassis cell Pseudomonas putida which does not have rapeseed oil utilization capacity is provided with high-efficiency rapeseed oil utilization capacity, so that the safe and high-efficiency degradation and production of rhamnolipid from a rapeseed oil carbon source system are realized.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and microbial metabolic engineering, specifically to recombinant Pseudomonas aeruginosa engineered strains that produce rhamnolipids, their construction methods, and applications. Background Technology

[0002] Rhamnolipids are a class of high-value-added biosurfactants. Utilizing inexpensive rapeseed oil as a carbon source for fermentation is an effective way to reduce industrial costs. However, the degradation of lipids by natural Pseudomonas aeruginosa is limited by the insufficient secretion efficiency of the extracellular lipase LipA and its folding chaperone LipH. Meanwhile, most wild-type Pseudomonas aeruginosa presents biosafety risks and low carbon source utilization during scale-up production, while safe chassis cells (such as Pseudomonas putridae KT2440), although capable of heterologously synthesizing rhamnolipids, exhibit extremely weak lipolysis capabilities. Summary of the Invention

[0003] The present invention aims to provide a recombinant Pseudomonas engineered strain that produces rhamnolipids, its construction method and application. This scheme can remove the metabolic bottleneck of Pseudomonas on rapeseed oil, and in particular, it can enable the safe chassis cells (Pseudomonas putidae) that cannot utilize rapeseed oil to achieve metabolic utilization of rapeseed oil.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A recombinant Pseudomonas engineered strain for producing rhamnolipids from rapeseed oil, wherein the recombinant Pseudomonas engineered strain is constructed by introducing recombinant plasmids into Pseudomonas as the host bacterium. The recombinant plasmid contains an arabinose-inducible promoter and a lipase gene regulated by the arabinose-inducible promoter. lipA and molecular chaperone lipH The lipA exist lipH Co-expressed in the host bacteria; The host bacteria are either *Pseudomonas aeruginosa* or engineered *Pseudomonas putida* strains that have integrated the rhamnosyl ester synthesis pathway.

[0005] In some embodiments of the present invention, the lipase and molecular chaperone are derived from Pseudomonas aeruginosa PAO1.

[0006] In some embodiments of the present invention, the *Pseudomonas aeruginosa* is *Pseudomonas aeruginosa* JLDR7-6Δpha, wherein JLDR7-6Δpha is derived from *Pseudomonas aeruginosa* JLDR7-6 as the starting strain, and is knocked out. phaC1 , phaC2 and phaD Gene acquisition.

[0007] In some embodiments of the present invention, the host bacterium is engineered Pseudomonas putidae KT114.

[0008] The present invention further provides a method for constructing the above-mentioned recombinant Pseudomonas engineered strain, the method comprising: Will lipA , lipH gene fragments, or lipA and lipH Serial segments lipAH The gene fragment was cloned into a linearized vector plasmid to construct a recombinant plasmid; Using Escherichia coli S17-1 as the donor bacterium for conjugation transfer, the constructed recombinant plasmid was introduced into the host bacterium through a biparental conjugation transfer method to construct a recombinant Pseudomonas engineered bacterium.

[0009] In some embodiments of the present invention, the vector plasmid is a pJN105 shuttle plasmid with an L-arabinose-inducible pBAD promoter.

[0010] The present invention further provides the application of the above-mentioned recombinant Pseudomonas engineered strain in the production of rhamnose esters.

[0011] In some embodiments of the present invention, the recombinant Pseudomonas engineered strain ferments rapeseed oil to produce rhamnose ester.

[0012] In some embodiments of the present invention, the recombinant Pseudomonas engineered strain is fermented and induced by adding 0.01-0.1% (w / v) L-arabinose.

[0013] In some embodiments of the present invention, when fermenting engineered recombinant Pseudomonas aeruginosa with Pseudomonas aeruginosa as the host bacterium, 0.05% (w / v) L-arabinose is added for induction. When fermenting recombinant Pseudomonas engineered strains with Pseudomonas putida as the host bacterium, 0.5% (g / L) L-arabinose is added for induction.

[0014] This invention constructs recombinant Pseudomonas engineered strains using either the self-selected Pseudomonas aeruginosa JLDR7-6Δpha with excellent characteristics or the safe chassis Pseudomonas putrefactive strain KT114 pre-integrated with the rhamnolipid synthesis pathway as chassis cells. During fermentation, precise induction of LipA and LipH co-expression through specific concentrations of arabinose enhances the rapeseed oil utilization capacity of Pseudomonas aeruginosa. In particular, it enables the safe chassis cell Pseudomonas putrefactive strains, which previously lacked rapeseed oil utilization capabilities, to possess highly efficient rapeseed oil utilization. The fermentation reaction exhibits low residual oil content, achieving safe and efficient degradation and production of rhamnolipids using rapeseed oil as a carbon source system, demonstrating significant industrial application value. Attached Figure Description

[0015] Figure 1It is the JLDR7-6Δpha engineered strain and JLDR7-6Δpha- in Example 2 lipAH A comparison of rhamnolipin yields during shake-flask fermentation using rapeseed oil as a carbon source by engineered bacteria under different concentrations of arabinose induction.

[0016] Figure 2 It is JLDR7-6Δpha- in Example 3 lipAH The growth curves of engineered bacteria using rapeseed oil as a carbon source and the fermentation results of rhamnolipin in a 5L fermenter.

[0017] Figure 3 It is the safe engineered bacterium KT114 based on *Pseudomonas putida* KT2440 in Example 4. lipAH Curves showing the growth and rhamnolipin fermentation results using rapeseed oil as the carbon source in a 5L fermenter. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0019] The general methods involved in the embodiments are described below: Product extraction, separation and determination method: After fermentation, the preferred steps for extracting and separating rhamnolipin include: collecting the fermentation broth and centrifuging at 8000 to 12000 rpm to remove the cells; adjusting the pH of the supernatant to 2.0 to 3.0 with hydrochloric acid for acid precipitation and letting it stand overnight; collecting the precipitate by centrifugation, extracting twice with ethyl acetate in equal volumes, combining the organic phases and concentrating to obtain crude rhamnolipin.

[0020] Method for determining rhamnolipin yield: The sulfuric acid-anthrone colorimetric method was used. The target analyte, after acid precipitation and organic solvent extraction, was extracted, the solvent was removed, and the mixture was brought to a final volume. The sulfuric acid-anthrone reagent was added, and the mixture was placed in a boiling water bath for color development. The absorbance was measured at 620 nm. A standard curve was constructed using L-rhamnose standards, and the total concentration of rhamnolipin in the fermentation broth was calculated accordingly.

[0021] Method for determining residual oil content in fermentation: Take an appropriate amount of fermentation broth or fermentation supernatant, add an equal volume of non-polar organic solvent (preferably n-hexane), and extract by vigorous shaking. After standing or centrifugation to separate the layers, collect the upper organic phase, and allow it to evaporate completely at room temperature to remove the organic solvent, obtaining the residual oily substance, which is then weighed. Calculate the concentration (g / L) of undegraded residual rapeseed oil in the fermentation system.

[0022] The host bacterium involved in the embodiment, Pseudomonas aeruginosa JLDR7-6Δpha, is based on JLDR7-6 (disclosed in the applicant's previous application CN116987654A), with the knockout strain... phaC1 (PA5056) phaC2 (PA5054) and phaD The (PA5053) gene was obtained and knocked out using the same method as the gene knockout method in CN116987654A.

[0023] The host bacterium involved in the embodiment is *Pseudomonas putida* KT114, which is an engineered bacterium *Pseudomonas putida* KT2440 that integrates the rhamnosyl ester synthesis pathway. Its construction method has been disclosed in the applicant's previous application CN121227611A.

[0024] The donor strain, E. coli S17-1, is a commercial strain and can be purchased from commercial channels.

[0025] Other biological materials and reagents are common commercial materials and reagents that can be purchased from commercial channels.

[0026] The following are definitions of some terms used in the embodiments: Conjugation transfer refers to the process by which plasmids are transferred to recipient cells through bacterial contact, relying on transfer-related genes (such as tra sites) carried by the plasmid itself or provided by helper strains.

[0027] Example 1: Construction of recombinant expression plasmid pJN105-lipAH and its conjugation and transfer procedure This embodiment details the complete technical process of constructing an expression vector using the broad-host shuttle plasmid pJN105 and introducing it into the recipient strain via parental conjugation transfer technology.

[0028] (1) Amplification of the target gene Genomic DNA was extracted from *Pseudomonas aeruginosa* JLDR7-6ΔPHA, a natural rhamnolipid-producing bacterium, using a bacterial genomic DNA extraction kit as a template for PCR amplification. Specific primers with restriction enzyme sites were designed based on the known sequence, and PCR amplification was performed using high-fidelity DNA polymerase to obtain a full-length DNA sample of approximately 2000 bp containing... lipA (The nucleotide sequence is shown in SEQ ID NO:1) and lipH (The tandem fragment of the nucleotide sequence shown in SEQ ID NO:2) lipAH (The nucleotide sequence is shown in SEQ ID NO:3). The PCR product was verified by 1% agarose gel electrophoresis and then purified using a gel extraction kit.

[0029] (2) Enzyme digestion and ligation of expression plasmids Recycle lipAH The fragment and the empty vector pJN105 plasmid were placed in the system respectively, and then... EcoRI and XbaIRestriction endonucleases were used for double digestion at 37°C for 2-4 hours. The digestion products were then purified and recovered by agarose gel electrophoresis. Subsequently, the digested pJN105 vector backbone was combined with… lipAH The target fragments were mixed at a molar ratio of 1:3, and DNA ligase was added to construct the recombinant expression plasmid pJN105-lipAH.

[0030] (3) Transformation and screening of donor Escherichia coli The ligation product was transformed into competent *E. coli* S17-1 cells containing the conjugation transfer element. The transformed bacterial culture was evenly spread on LB agar plates containing 20 μg / mL gentamicin and incubated upside down at 37°C for 12–16 hours. Single colonies were picked from the plates and verified by colony PCR and double enzyme digestion of plasmids. Positive clones were sent to a sequencing company for sequencing comparison. Clones with 100% correct sequencing results were those containing pJN105- lipAH The donor bacterium is E. coli S17-1.

[0031] (4) Introduction of Pseudomonas aeruginosa via parental conjugation transfer method The donor bacteria E. coli S17-1 (carrying pJN105-) lipAH The donor bacteria JLDR7-6Δpha and recipient bacteria were inoculated into LB liquid medium (20 μg / mL gentamicin was added to the donor bacteria) and cultured at 37°C with shaking until the logarithmic growth phase (OD600 approximately 0.6-0.8).

[0032] 1) Centrifugation and washing: Take 1 mL of donor bacteria and recipient bacteria respectively, centrifuge to collect the bacterial cells, and wash twice with sterile physiological saline or LB medium to completely remove antibiotics.

[0033] 2) Mixed incubation: Mix donor and recipient bacteria at a 1:1 ratio and resuspend in 50 μL of liquid. Add the mixture dropwise to the center of an antibiotic-free LB agar plate. After the bacterial suspension has air-dried, incubate at 37°C for 6-8 hours to allow plasmid transfer to complete.

[0034] 3) Elution and Screening: After incubation, the bacterial growth on the plate was eluted with 1 mL of sterile physiological saline and thoroughly dispersed. The elution buffer was serially diluted and spread onto dual-selectivity plates containing 50 μg / mL gentamicin and 20 μg / mL triclosan. After incubation at 37℃ for 16-24 hours, transformants grown on the plates were picked for PCR verification, ultimately successfully obtaining the recombinant engineered strain JLDR7-6Δpha- lipAH .

[0035] The method for introducing plasmids using KT114 strain as the recipient is the same, using the recombinant plasmid pJN105- lipAHUsing *Escherichia coli* S17-1 as the donor bacterium and KT114 as the recipient bacterium, the recombinant plasmid was introduced into KT114 via a parental conjugation transfer method. After verification through antibiotic resistance screening, the recombinant engineered bacterium KT114- was successfully obtained. lipAH .

[0036] Example 2: Construction of expression plasmid and comparison of the effects of different concentrations of arabinose on JLDR7-6Δpha fermentation (1) Plasmid construction and conjugation transfer introduction Using the genome of self-selected Pseudomonas aeruginosa JLDR7-6Δpha as a template, PCR amplification was performed on tandem sequences containing upstream and downstream restriction enzyme sites. lipAH (The nucleotide sequence is shown in SEQ ID NO:3). The pJN105 plasmid and the target fragment were double-digested with EcoRI and XbaI, and ligated using T4 DNA ligase to obtain the recombinant plasmid pJN105- lipAH The recombinant plasmid was first transformed into Escherichia coli S17-1 (… E. coli Positive transformants were obtained in S17-1); subsequently, using pJN105- lipAH Using S17-1 as the donor strain and JLDR7-6Δpha as the recipient strain, biparental conjugation transfer was performed. After mixing the strains, they were incubated on antibiotic-free plates for conjugation. The resulting mixture was then eluted and plated onto plates containing selective antibiotics such as gentamicin for screening and verification. The recombinant engineered strain JLDR7-6Δpha was successfully obtained. lipAH .

[0037] (2) Gradient fermentation of arabinose concentration in shake flasks JLDR7-6Δpha- lipAH The strain was inoculated into a 250 mL shake flask (working volume 50 mL) and fermented using rapeseed oil as the carbon source. The fermentation medium formula was: rapeseed oil 30 g / L, NaNO3 6 g / L, yeast extract 3 g / L, KH2PO4 1 g / L, Na2HPO4 1 g / L, CaCl2·2H2O 0.1 g / L, MgSO4 0.1 g / L, pH 7.0. It was cultured at 37℃ and 200 rpm.

[0038] (3) Induced expression When the bacterial cells grew to an OD600 of 0.8, the shake flasks were divided into three groups, and L-arabinose with final concentrations of 0%, 0.05%, and 0.5% w / v was added to the system for induction (i.e., 0, 0.5 g / L, and 5 g / L).

[0039] (4) Result evaluation (e.g.) Figure 1 (As shown) Using the aforementioned method for determining rhamnolipin yield, the rhamnolipin yield was measured after 72 hours of fermentation.

[0040] 1) 0% induction group: Due to the lack of inducer, the pBAD promoter is in the off state. lipAH It is not expressed at all, and the rhamnolipid production measured by the sulfuric acid-anthrone method is lower than that of the host bacterium (JLDR7-6Δpha).

[0041] 2) 0.5% high concentration induction group: Strong induction led to overexpression of exogenous proteins, which brought a huge metabolic burden to the host cells of Pseudomonas aeruginosa and even produced plasmid toxicity. Cell biomass decreased and rhamnolipid production decreased, failing to reach the optimal production capacity, but the production was higher than that of the host bacteria with the same induction concentration.

[0042] 3) 0.05% Optimal Induction Concentration Group: This concentration achieved a perfect balance between LipA / LipH protein expression and JLDR7-6Δpha cell growth, with lipase being secreted into the extracellular space moderately and efficiently. The fermentation broth was in a completely homogeneous emulsion state, rapeseed oil was extremely consumed, and the total yield of rhamnolipids increased to 21.5 g / L. This example clarifies that 0.05% is the optimal induction concentration for fermentation production using engineered Pseudomonas aeruginosa strains.

[0043] In addition, comparing the host bacteria and JLDR7-6Δpha- lipAH The fermentation results show that the host bacteria itself has the ability to utilize rapeseed oil, and the optimal concentration is JLDR7-6Δpha- lipAH The yield of the bacteria was better than that of the host bacteria, but the increase was not significant.

[0044] Example 3 JLDR7-6Δpha- lipAH Scale-up fermentation of engineered bacteria in a 5L fermenter JLDR7-6Δpha- lipAH The seed culture was inoculated into a 5L fermenter (working volume 2L) at a 5% inoculation rate. 60g / L rapeseed oil was used as the carbon source (rapeseed oil 60g / L, NaNO3 6g / L, yeast extract 3g / L, KH2PO4 1g / L, Na2HPO4 1g / L, CaCl2·2H2O 0.1g / L, MgSO4 0.1g / L, pH 7.0). The culture temperature was 37℃, and dissolved oxygen was maintained above 30%.

[0045] When fermentation reached the logarithmic phase, based on the optimization results of Example 2, a single-pass feed of L-arabinose at a final concentration of 0.05% induced fermentation. lipAH The expression is fermented for a total of 5 days.

[0046] The results are as follows Figure 2As shown, with sufficient oxygen supply in the fermenter and induction at the optimal concentration of 0.05%, the JLDR7-6Δpha- precipitate was determined by the sulfuric acid-anthrone method. lipAH The total rhamnolipid yield exceeds 40 g / L.

[0047] Example 4 KT114- lipAH Scale-up fermentation of engineered bacteria in a 5L fermenter A 5L fermenter was used with 50g / L rapeseed oil as the carbon source (50g / L rapeseed oil, 6g / L NaNO3, 3g / L yeast extract, 1g / L KH2PO4, 1g / L Na2HPO4, 0.1g / L CaCl2·2H2O, 0.1g / L MgSO4, pH 7.0), and the fermentation temperature was set at 30℃. During the logarithmic phase, 0.5% L-arabinose of *Pseudomonas putida* was added for induction, and fermentation lasted for 5 days.

[0048] The results are as follows Figure 3 As shown, KT2440 and KT114 themselves do not have the ability to utilize rapeseed oil, while the engineered bacteria KT114- lipAH Under high-concentration induction with 0.5% arabinose, after 5 days of cultivation, the rapeseed oil in the system was almost completely consumed, and rhamnolipin accumulated to 4.1 g / L. This means that, after modification, KT114, which originally lacked the ability to utilize rapeseed oil, can achieve efficient utilization of rapeseed oil under specific concentrations of arabinose induction. This invention provides a modified strain that can utilize heterologous safe chassis bacteria and use rapeseed oil as a carbon source to produce rhamnolipin, showing promising prospects for industrial application.

Claims

1. A recombinant Pseudomonas engineered strain for producing rhamnolipids from rapeseed oil, characterized in that, The recombinant Pseudomonas engineered strain was constructed by introducing recombinant plasmids into Pseudomonas as the host strain. The recombinant plasmid contains an arabinose-inducible promoter and a lipase gene regulated by the arabinose-inducible promoter. lipA and molecular chaperone lipH The lipA exist lipH Co-expressed in the host bacteria; The host bacteria are either *Pseudomonas aeruginosa* or engineered *Pseudomonas putida* strains that have integrated the rhamnosyl ester synthesis pathway.

2. The recombinant Pseudomonas engineered strain according to claim 1, characterized in that, The lipase and molecular chaperone were derived from Pseudomonas aeruginosa PAO1.

3. The recombinant Pseudomonas engineered strain according to claim 1, characterized in that, The *Pseudomonas aeruginosa* strain mentioned is *Pseudomonas aeruginosa* JLDR7-6Δpha, which is derived from *Pseudomonas aeruginosa* strain JLDR7-6 by knockout. phaC1 , phaC2 and phaD Gene acquisition.

4. The recombinant Pseudomonas engineered strain according to claim 1, characterized in that, The host bacterium is engineered Pseudomonas putidae strain KT114.

5. The method for constructing the recombinant Pseudomonas engineered strain according to any one of claims 1 to 4, characterized in that, The construction method includes: Will lipA , lipH gene fragments, or lipA and lipH Serial segments lipAH The gene fragment was cloned into a linearized vector plasmid to construct a recombinant plasmid; Using Escherichia coli S17-1 as the donor bacterium for conjugation transfer, the constructed recombinant plasmid was introduced into the host bacterium through a biparental conjugation transfer method to construct a recombinant Pseudomonas engineered bacterium.

6. The construction method according to claim 5, characterized in that, The vector plasmid is the pJN105 shuttle plasmid with an L-arabinose-inducible pBAD promoter.

7. The use of the recombinant Pseudomonas engineered strain according to any one of claims 1 to 5 in the production of rhamnosyl esters.

8. The application according to claim 7, characterized in that, The recombinant Pseudomonas engineered strain ferments rapeseed oil to produce rhamnose ester.

9. The application according to claim 7, characterized in that, The recombinant Pseudomonas engineered strain was fermented and induced by adding 0.01-0.1% (w / v) L-arabinose.

10. The application according to claim 9, characterized in that, When fermenting recombinant Pseudomonas aeruginosa engineered strains with Pseudomonas aeruginosa as the host bacterium, 0.05% (w / v) L-arabinose was added for induction. When fermenting engineered recombinant Pseudomonas bacteria with Pseudomonas putida as the host, 0.5% w / v L-arabinose was added for induction.

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