Pseudomonas aeruginosa engineering bacterium as well as construction method and application thereof

Abstract

The invention provides a pseudomonas aeruginosa engineering bacterium, which is based on a PAO1 wild type strain, through combined knockout of three groups of functional genes of virulence factor and biofilm polysaccharide, the toxicity and biofilm capability are synergistically reduced, meanwhile, the synthesis output of rhamnolipid is improved, compared with single path modification, the toxicity-related phenotype is remarkably reduced, and the application safety is improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered strain of Pseudomonas aeruginosa, a method for constructing the engineered strain of Pseudomonas aeruginosa, and the application of the engineered strain of Pseudomonas aeruginosa in the preparation of rhamnolipids. Background Technology

[0002] Pseudomonas aeruginosa PAO1 is an important rhamnolipin-producing strain. However, its acute virulence and biofilm phenotype limit the safety, controllability, and production stability of its industrial applications. The type III secretion system (T3SS) effector proteins ExoS / ExoT are known to be closely associated with host cell damage and acute toxicity; meanwhile, PSL and PEL extracellular polysaccharides (encoded by the psl and pel gene clusters, respectively) are major matrix components of biofilms, promoting adhesion.

[0003] During fermentation, biofilm formation causes microbial cells to adhere to solid surfaces such as fermenters and pipelines, creating dead zones on the walls and hindering mass transfer. This results in uneven distribution of dissolved oxygen and substrate, leading to fermentation fluctuations, local instability, and poor batch-to-batch consistency. Therefore, strategies that solely rely on increasing metabolic flux often fail to resolve the complex contradictions of "safety-stability-yield" in engineering applications.

[0004] Therefore, this invention implements synergistic knockout of biofilm polysaccharide-related genes pelA and pslBCD, aiming to inhibit biofilm formation during fermentation, reduce wall adhesion, improve mass transfer conditions and the homogeneity of the culture system, thereby enhancing fermentation stability and yield performance. Summary of the Invention

[0005] The primary objective of this invention is to provide an engineered strain of Pseudomonas aeruginosa to address the shortcomings of existing technologies, such as the poor fermentation stability of Pseudomonas aeruginosa.

[0006] A second objective of this invention is to provide a method for constructing engineered Pseudomonas aeruginosa bacteria.

[0007] A third objective of this invention is to provide an engineered strain of Pseudomonas aeruginosa for the preparation of rhamnolipids.

[0008] To achieve the above objectives, the following technical solution is adopted: An engineered strain of *Pseudomonas aeruginosa*, based on the PAO1 wild-type strain, has at least two of the following three gene groups knocked out in its genome: Group I: exoS and exoT; Group II: One or more of pslB, pslC, and pslD; Group III: pelA.

[0009] In this invention, genome group I has been knocked out, and at least one of genome groups II and III has also been knocked out: Group I: exoS and exoT; Group II: one or more of pslB, pslC, and pslD; Group III: pelA.

[0010] In this invention, genome group I has been knocked out, and at least one of genome groups II and III has also been knocked out: Group I: exoS and exoT; Group II: pslB, pslC, pslD; Group III: pelA.

[0011] In some embodiments of the present invention, the following genes have been knocked out in the genome: exoS, exoT, pslB, pslC, and pslD.

[0012] In some embodiments of the present invention, the following genes have been knocked out in the genome: exoS, exoT, pslB, pslC, pslD, and pelA.

[0013] In some embodiments of the present invention, the following genes have been knocked out in the genome: exoS, exoT, and pelA.

[0014] A method for constructing engineered Pseudomonas aeruginosa bacteria includes the following steps: using the CRISPR-Cas9 system (CRISPR-associated protein 9), a guide RNA (gRNA) and homologous recombination fragments targeting the target site are designed to complete site-specific knockout; engineered Pseudomonas aeruginosa bacteria are obtained through selective culture screening, molecular biology verification, and finally sequencing confirmation.

[0015] Application of the above-mentioned engineered Pseudomonas aeruginosa strain in the preparation of rhamnolipids.

[0016] The present invention has the following beneficial effects: (1) The Pseudomonas aeruginosa engineered strain of the present invention reduces toxicity and biofilm capacity by knocking out three functional genes: “virulence factor + biofilm polysaccharide”, while increasing the synthesis output of rhamnolipids. Compared with single-pathway modification, it significantly reduces toxicity-related phenotypes and improves application safety.

[0017] (2) The engineered Pseudomonas aeruginosa strain of this invention exhibits superior rhamnolipid synthesis throughput, significantly improved relative yield index, and better batch-to-batch stability under a microenvironment with reduced biofilm and toxicity stress. It does not rely on a specific culture system or a single detection method, but uses relative quantitative indicators as proof of effectiveness, making it suitable for scale-up in different processes.

[0018] (3) In this invention, ΔpslBCD and ΔpelA weaken the biofilm matrix, reduce adhesion to the fermenter and pipeline walls, alleviate uneven distribution of dissolved oxygen and substrate, reduce batch fluctuations, improve fermentation controllability, and make the fermentation process more user-friendly. The combination of ΔpslBCD, ΔpelA and ΔexoST gene knockout has a synergistic effect on yield improvement, with unexpected results. Attached Figure Description

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 This is the pACRISPR-exoS map constructed in this invention; Figure 2 This is the pACRISPR-exoT plasmid map constructed in this invention; Figure 3 This is the pACRISPR-pslBCD map of the plasmid constructed in this invention; Figure 4 This is the PCRISPR-pelA pattern of the plasmid constructed in this invention; Figure 5 This is the LDH cytotoxicity assay of ΔexoST engineered bacteria on A549 cells; Figure 6 This is a comparison of the LDH cytotoxicity test results of ΔexoST engineered bacteria on A549 cells; Figure 7 This is a comparison chart of the relative yields of rhamnolipin in different genotype strains of this invention (including wild type, ΔpelA, ΔpslBCD, and ΔpslBCD-ΔpelA). Figure 8 This is a comparison chart of the relative yields of rhamnolipin in different genotype strains of this invention (including wild type, ΔexoS, ΔexoT, and ΔexoST). Figure 9 This is a comparison chart of the relative yields of rhamnolipin in different genotype strains of this invention (including wild type, ΔpslBCD-ΔexoST, ΔpslBCD-ΔpelA-ΔexoST). Detailed Implementation

[0021] This invention provides a method for constructing engineered *Pseudomonas aeruginosa* bacteria. The method employs the CRISPR-Cas9 system, featuring clustered regularly spaced short palindromic repeats, and includes the design of guide RNA (gRNA) and homologous recombination fragments targeting specific sites for site-directed knockout. Through selective culture screening, molecular biology verification, and final sequencing confirmation, engineered *Pseudomonas aeruginosa* bacteria are obtained. The specific steps include: A. Target site design and grouping: Design editing sites and homologous recombination strategies for exoS, exoT, pslB / pslC / pslD, and pelA; select single genes, subsets, or gene clusters (such as pslBCD) for knockout as needed.

[0022] B. Vector System Construction: Construct an editing vector containing guide ribonucleic acid and homologous recombination fragments, and configure a vector system for expressing CRISPR-related protein 9. The vector can be a functional equivalent of an existing CRISPR vector (such as "pCRISPR-like vector" or "Cas9 expression vector"), and the specific name can be replaced with an equivalent tool vector.

[0023] C. Transformation and screening: The vector is sequentially or co-introduced into the host strain, and the culture medium is used to screen for positive clones by using selection markers that match the vector.

[0024] D. Molecular validation: Fragment amplification of the target site is performed using polymerase chain reaction, and the expected deletion or substitution event is confirmed by sequencing.

[0025] E. Vector removal and stability verification: Vector loss was achieved through conventional anti-selection strategies (e.g., sucrose-based anti-selection marker systems) to obtain stable engineered strains without exogenous vectors; key sites were re-sequencing.

[0026] The effects of different gene knockouts on engineered Pseudomonas aeruginosa strains in this invention are as follows: 1. The effect of exoST gene knockout (T3SS-related gene) on virulence: exoST typically refers to effector genes in the type III secretion system (T3SS) of *Pseudomonas aeruginosa*, such as exoS, exoT, exoU, and exoY. T3SS is a key mechanism for *P. aeruginosa* to secrete virulence proteins into host cells and plays an important role in various infections. The cAMP-Vfr system is a crucial regulator of T3SS expression.12 Vfr directly activates the transcription of ExsA, which is the core activator of T3SS gene expression. NrtR (PA4916) has also been shown to affect T3SS expression and the pathogenicity of *P. aeruginosa* in a mouse model of acute pneumonia through the cAMP / Vfr pathway. Studies have shown that overexpression of the T3SS central activator ExsA or exogenous cAMP supplementation can restore T3SS expression in the ΔnrtR mutant. Furthermore, Hfq and sRNA 179 have been found to inhibit the expression of cAMP-Vfr and T3SS regulators, further confirming the importance of T3SS in the virulence regulatory network. Although the provided literature does not directly state specific data indicating that knocking out a particular exoST gene leads to reduced virulence, it is generally believed that the absence of T3SS effector factors significantly weakens bacterial pathogenicity. For example, many studies consider T3SS effector factors as important virulence determinants, and their loss of function is usually accompanied by reduced virulence. Therefore, it can be inferred that knocking out the exoST gene reduces the virulence of *Pseudomonas aeruginosa*.

[0027] 2. The impact of pelA and pslBCD gene knockout (biofilm-related genes) on fermentation process stability and yield: The pelA and pslBCD genes are closely related to biofilm formation in *Pseudomonas aeruginosa*. The pel gene cluster is responsible for producing the dextran biofilm matrix, while the psl gene cluster encodes glycans, a major component of the biofilm. Biofilms play a crucial role in chronic *Pseudomonas aeruginosa* infection, protecting bacteria from the host immune system and antibiotics. pslBCD and pelA inhibit biofilm formation, making it less likely for bacteria to adhere to the fermentation wall during fermentation. Knocking out pslBCD and pelA weakens biofilm formation and wall adhesion during fermentation, significantly improving mass transfer and homogeneity, resulting in stable and high-performing rhamnolipid-producing strains with better process compatibility and yield performance during fermentation.

[0028] Example 1 ΔexoST double gene knockout strain and cytotoxicity verification First, a ΔexoS / ΔexoT single-gene defective strain was constructed. Then, using the same method, iterative gene knockout was performed based on the single-gene defective strain. The following section uses the construction of the ΔexoT single-gene knockout strain as an example.

[0029] I. Construction of plasmid pCRISPR-exoT 1.1 Obtaining the Homologous Arm Using the PAO1 genome as a template, approximately 500 bp homologous arm fragments (exoT-up and exoT-dn) upstream and downstream of the exoT gene were amplified by PCR.

[0030] Primer design: The exoT-up reverse primer and the exoT-dn forward primer each carry a 20-25bp complementary sequence, which are used for overlap PCR fusion to obtain the exoT-up-dn fragment (about 1000bp).

[0031] 1.2 Obtaining gRNA fragments gRNA design: A suitable PAM sequence (5'-NGG-3') was selected from the coding region of the exoT gene to design a 20nt target sequence (N20). The specificity of the target sequence was verified by BLAST alignment of the PAO1 genome.

[0032] Amplification: The N20-gRNA fragment was amplified using primers containing the N20 sequence.

[0033] 1.3 Plasmid Assembly Use Gibson Assembly to connect the following three fragments: Linearized pCRISPR vector exoT-up-dn homologous arm fragment N20-gRNA fragment Reaction conditions: 50°C, 1 hour.

[0034] 1.4 Plasmid Validation The ligation product was transformed into competent *E. coli* cells (e.g., DH5α) and screened on plates containing the appropriate antibiotics. Positive clones were picked, and plasmids were extracted. The correctness of the homologous arm sequences, gRNA sequences, and vector backbone was verified by sequencing. Plasmids with correct sequencing results were named pCRISPR-exoT, such as... Figure 2 As shown, (constructing pCRISPR-exoS as follows) Figure 1 (As shown).

[0035] II. CRISPR-Cas9 gene knockout 2.1 pCasPA plasmid introduction The plasmid pCasPA containing the Cas9 gene was electroporated into the PAO1 strain, and positive clones were screened on LB plates containing 100 µg / mL tetracycline. The presence of pCasPA was verified by colony PCR or plasmid extraction to obtain the PAO1-pCasPA strain.

[0036] 2.2 Cas9 protein induced expression Select PAO1-pCasPA single clones and inoculate them into LB medium containing 50 µg / mL tetracycline, and culture overnight at 37°C. The next day, dilute the medium 1:50 to fresh LB medium, add 0.02% L-arabinose to induce Cas9 expression, and continue culturing for 2-4 hours.

[0037] 2.3 Importing Edited Plasmids Collect the induced bacterial culture and prepare electroporation competent cells. Electroporate the plasmid pCRISPR-exoT (≥1 µg) and plate it on LB agar plates containing 100 µg / mL tetracycline and 150 µg / mL carbenicillin. Incubate at 37°C for 48–72 hours.

[0038] III. Gene Knockout Identification and Plasmid Elimination 3.1 Preliminary PCR identification Single clones were selected for colony PCR identification.

[0039] Identification primer design: The upstream primer is located on the genomic sequence outside the exoT-up homologous arm, and the downstream primer is located on the genomic sequence outside the exoT-dn homologous arm.

[0040] Expected results: Wild-type PAO1: The amplification product is approximately 1000-1500 bp (containing the complete exoT gene). ΔcyaA mutant strain: The amplification product is about 200-300bp (the exoT gene is deleted, and only the homologous arm linker region is retained). Positive clones that produce short fragments are selected for subsequent steps.

[0041] 3.2 Plasmid elimination Clones identified by PCR were streaked onto LB agar plates containing 15% (w / v) sucrose and free of NaCl (NSLB plates) and incubated overnight at 37°C. Both pCasPA and pCRISPR vectors contain the sacB gene, the expression product of which is lethal to bacteria in the presence of sucrose, allowing for reverse selection of strains that have lost plasmids.

[0042] 3.3 Plasmid loss verification Pick single colonies from NSLB plates and spot them onto the following culture media: LB tablets (antibiotic-free) LB + 100 µg / mL tetracycline plate LB + 150 µg / mL carbenicillin plate A strain that has successfully eliminated the plasmid is one that grows only on antibiotic-free LB plates.

[0043] 3.4 Final Verification The plasmid-eliminated strain was validated again by colony PCR to confirm the stable existence of the gene knockout. If necessary, whole-genome sequencing can be performed to confirm precise deletion of the exoT gene without off-target effects. The strain whose sequencing verification is correct is the PAO1-ΔexoT engineered strain.

[0044] IV. Cytotoxicity assay (LDH release method) A549 cells were seeded in 96-well plates at approximately 1×10^4 cells / well and cultured at 37°C with 5% CO2 for 24 h. ΔexoST and the non-knockout control strain were cultured to the logarithmic growth phase, washed with PBS, and resuspended in McCoy's 5A medium. Infected cells at a bacterial:cell ratio of ≈50:1 for 24 h, supernatant was collected and quantified using an LDH detection kit; cells treated with lysis buffer served as a positive control for maximum release, and untreated cells served as a background control. Results: The ΔexoST strain released significantly less LDH from A549 cells than the non-knockout control (see [link]). Figure 5 , Figure 6 This indicates a significant reduction in toxicity.

[0045] Production evaluation: Fermentation was conducted under the same culture and fermentation conditions as the PAO1 wild-type. Rhamnolipids in the samples were quantitatively analyzed using liquid chromatography-mass spectrometry (LC / MS). The relative yield of rhamnolipids was calculated by comparing the mass spectrometric signal intensity of the wild-type control group with the signal intensity ratio. Compared with the PAO1 wild-type, the relative yield index of rhamnolipids in the ΔexoS, ΔexoT, and ΔexoST double-gene knockout engineered bacteria did not increase (see...). Figure 8 ).

[0046] Example 2 ΔpslBCD three-gene knockout strain.

[0047] Iterative gene knockout was performed using the same method as in Example 1 to construct a ΔpslBCD three-gene knockout strain. The pACRISPR-pslBCD plasmid map constructed during the process is shown below. Figure 3 As shown.

[0048] Production evaluation: Fermentation was conducted under the same culture and fermentation conditions as the PAO1 wild type, and the relative yield of rhamnolipin was calculated using the yield detection method described in Example 1. Compared to the PAO1 wild type, the relative yield index of rhamnolipin in the ΔpslBCD gene knockout engineered strain was not increased (see Example 1). Figure 7 ).

[0049] Example 3 ΔpelA gene knockout strain.

[0050] Gene knockout was performed using the same method as in Example 1 to construct a ΔpelA gene knockout strain. The PCRISPR-pelA map of the plasmid constructed during the process is shown below. Figure 4 As shown.

[0051] Production evaluation: Fermentation was conducted under the same culture and fermentation conditions as the PAO1 wild type, and the relative yield of rhamnolipin was calculated using the yield detection method described in Example 1. Compared to the PAO1 wild type, the relative yield index of rhamnolipin in the ΔpelA gene knockout engineered strain was not increased (see Example 1). Figure 7 ).

[0052] Example 4 ΔpslBCD-ΔpelA gene knockout strain.

[0053] Gene knockout was performed using the same method as in Example 1 to construct the ΔpslBCD-ΔpelA gene knockout strain.

[0054] Production evaluation: Fermentation was conducted under the same culture and fermentation conditions as the PAO1 wild type, and the relative yield of rhamnolipin was calculated using the yield detection method described in Example 1. Compared to the PAO1 wild type, the relative yield index of rhamnolipin in the ΔpslBCD-ΔpelA gene knockout engineered strain was not increased (see Example 1). Figure 7 ).

[0055] Example 5 ΔpslBCD-ΔexoST gene knockout strain.

[0056] The same method as in Example 1 was used to perform iterative gene knockout to construct the ΔpslBCD-ΔexoST gene knockout strain.

[0057] Production evaluation: Fermentation was conducted under the same culture and fermentation conditions as the PAO1 wild type, and the relative yield of rhamnolipin was calculated using the yield detection method described in Example 1. Compared to the PAO1 wild type, the relative yield index of rhamnolipin in the ΔpslBCD-ΔexoST gene knockout engineered strain was increased by 1.85 times (see Example 1). Figure 9 The yields of the ΔpslBCD and ΔexoST gene knockout strains did not increase, demonstrating that the ΔpslBCD-ΔexoST gene knockout has a synergistic effect on yield improvement and has an unexpected effect.

[0058] Example 6 ΔpslBCD-ΔpelA-ΔexoST gene knockout strain.

[0059] The same method as in Example 1 was used to perform iterative gene knockout to construct the ΔpslBCD-ΔpelA-ΔexoST gene knockout strain.

[0060] Production evaluation: Fermentation was carried out under the same culture and fermentation conditions as the PAO1 wild type, and the relative yield of rhamnolipin was calculated using the yield detection method described in Example 1. Compared with the PAO1 wild type, the relative yield index of rhamnolipin in the ΔpslBCD-ΔpelA-ΔexoST gene knockout engineered strain was increased by 1.67 times (see Example 1). Figure 9 ).

[0061] The preferred gene knockout construction sequence of this invention is to first perform ΔexoST to preferentially reduce toxicity, followed by ΔpslBCD and ΔpelA editing. This invention utilizes an engineering pathway of "first attenuating toxicity (ΔexoST) – then weakening the membrane (ΔpslBCD / ΔpelA)," which, while ensuring safety, reduces biofilm formation and wall adhesion during fermentation, significantly improving mass transfer and homogeneity, resulting in stable and high-performing rhamnolipid-producing strains. Compared to wild-type and single-point modification, the ΔpslBCD-ΔexoST and ΔpslBCD-ΔpelA-ΔexoST combinations demonstrate better process compatibility and yield performance in small-scale fermentation.

[0062] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. An engineered strain of *Pseudomonas aeruginosa*, characterized in that, Based on the PAO1 wild-type strain, at least two of the following three gene groups have been knocked out in the genome: Group I: exoS and exoT; Group II: One or more of pslB, pslC, and pslD; Group III: pelA.

2. The engineered Pseudomonas aeruginosa strain according to claim 1, characterized in that, The genome has had genome group I knocked out, and at least one of genome groups II and III has also been knocked out: Group I: exoS and exoT; Group II: One or more of pslB, pslC, and pslD; Group III: pelA.

3. The engineered Pseudomonas aeruginosa strain according to claim 2, characterized in that, The genome has already had genomic group I knocked out, and at least one of genomic groups II and III has also been knocked out: Group I: exoS and exoT; Group II: pslB, pslC, pslD; Group III: pelA.

4. The engineered Pseudomonas aeruginosa strain according to claim 3, characterized in that, The following genes have been knocked out in the genome: exoS, exoT, pslB, pslC, and pslD.

5. The engineered Pseudomonas aeruginosa strain according to claim 4, characterized in that, The following genes have been knocked out in the genome: exoS, exoT, pslB, pslC, pslD, and pelA.

6. The engineered Pseudomonas aeruginosa strain according to claim 3, characterized in that, The following genes have been knocked out in the genome: exoS, exoT, and pelA.

7. A method for constructing engineered Pseudomonas aeruginosa bacteria according to any one of claims 1-6, characterized in that, Includes the following steps: The CRISPR-Cas9 system, characterized by clustered regular-spaced short palindromic repeats, was used to design guide RNAs (gRNAs) and homologous recombination fragments targeting specific sites for targeted knockout. The engineered strain of Pseudomonas aeruginosa was obtained through selective culture screening, molecular biology verification, and finally sequencing confirmation.

8. The use of an engineered strain of Pseudomonas aeruginosa as described in any one of claims 1-6 in the preparation of rhamnolipids.

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