Improvement of pyoluteorin production in Pseudomonas protegens H78 through engineering its biosynthetic and regulatory pathways
Pyoluteorin (Plt) is a PKS-NRPS hybrid antibiotic that is produced by Pseudomonas spp. and shows strong antifungal and antibacterial activities. Pseudomonas protegens H78, which was isolated from the rape rhizosphere in Shanghai, can produce a large array of secondary metabolites, including antibiotics and siderophores. Plt is produced at low levels in the H78 wild-type strain. This study aimed to improve Plt production through combinatory genetic engineered strategies. Plt production was significantly enhanced (by14.3-fold) in the strain engineered by the following steps: (1) deletion of the translational repressor gene rsmE in the Gac/Rsm-RsmE pathway; (2) deletion of the ATP-dependent protease gene lon that encodes a potential enzyme that degrades positive regulators; (3) deletion of the negative regulatory gene pltZ of the Plt ABC-type transporter operon pltIJKNOP; (4) deletion of an inhibitory sequence within the operator of the transcriptional activator gene pltR; and (5) overexpression of the pltIJKNOP transport operon. The Plt production of the final engineered strain was increased to 214 from 15 μg ml−1 in the H78 wild-type strain. In addition, the pltA gene in the pltLABCDEFG biosynthetic operon was characterized as the gene encoding the rate-limiting enzyme in the Plt biosynthetic pathway of H78. However, overexpression of the rate-limiting enzyme gene pltA or the transcriptional activator gene pltR did not further improve Plt biosynthesis in the above multiple-gene knockout strains.
KeywordsPlant growth-promoting rhizobacteria Pseudomonas Antibiotic biosynthesis Genetic engineering Metabolic engineering
This study was supported by the National Natural Science Foundation of China (31470196 and 31270083 to Xianqing Huang).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants by any of the authors.
- Haas D, Keel C (2003) Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease. Annu Rev Phytopathol 41:117–153. https://doi.org/10.1146/annurev.phyto.41.052002.095656 CrossRefPubMedGoogle Scholar
- Jousset A, Schuldes J, Keel C, Maurhofer M, Daniel R, Scheu S, Thuermer A (2014) Full-genome sequence of the plant growth-promoting bacterium Pseudomonas protegens CHA0. Genome Announc 2(2):e00322–14 https://doi.org/10.1128/genomeA.00322-14
- Liu Y, Shi H, Wang Z, Huang X, Zhang X (2018) Pleiotropic control of antibiotic biosynthesis, flagellar operon expression, biofilm formation, and carbon source utilization by RpoN in Pseudomonas protegens H78. Appl Microbiol Biotechnol 102:9719–9730. https://doi.org/10.1007/s00253-018-9282-0 CrossRefPubMedGoogle Scholar
- Lu J, Huang X, Li K, Li S, Zhang M, Wang Y, Jiang H, Xu Y (2009) LysR family transcriptional regulator PqsR as repressor of pyoluteorin biosynthesis and activator of phenazine-1-carboxylic acid biosynthesis in Pseudomonas sp. M18. J Biotechnol 143(1):1–9. https://doi.org/10.1016/j.jbiotec.2009.06.008 CrossRefPubMedGoogle Scholar
- Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GS, Mavrodi DV, DeBoy RT, Seshadri R, Ren Q, Madupu R, Dodson RJ, Durkin AS, Brinkac LM, Daugherty SC, Sullivan SA, Rosovitz MJ, Gwinn ML, Zhou L, Schneider DJ, Cartinhour SW, Nelson WC, Weidman J, Watkins K, Tran K, Khouri H, Pierson EA, Pierson LS 3rd, Thomashow LS, Loper JE (2005) Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat Biotechnol 23(7):873–878. https://doi.org/10.1038/nbt1110 CrossRefPubMedGoogle Scholar
- Pechy-Tarr M, Bottiglieri M, Mathys S, Lejbolle KB, Schnider-Keel U, Maurhofer M, Keel C (2005) RpoN (σ 54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0. Molecular plant-microbe interactions. MPMI 18(3):260–272. https://doi.org/10.1094/MPMI-18-0260 CrossRefPubMedGoogle Scholar
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
- Takeuchi K, Tsuchiya W, Noda N, Suzuki R, Yamazaki T, Haas D (2014) Lon protease negatively affects GacA protein stability and expression of the Gac/Rsm signal transduction pathway in Pseudomonas protegens. Environ Microbiol 16(8):2538–2549. https://doi.org/10.1111/1462-2920.12394 CrossRefPubMedGoogle Scholar
- Vincent F, Round A, Reynaud A, Bordi C, Filloux A, Bourne Y (2010) Distinct oligomeric forms of the Pseudomonas aeruginosa RetS sensor domain modulate accessibility to the ligand binding site. Environ Microbiol 12(6):1775–1786. https://doi.org/10.1111/j.1462-2920.2010.02264.x CrossRefPubMedGoogle Scholar
- Winstanley C, Langille MG, Fothergill JL, Kukavica-Ibrulj I, Paradis-Bleau C, Sanschagrin F, Thomson NR, Winsor GL, Quail MA, Lennard N, Bignell A, Clarke L, Seeger K, Saunders D, Harris D, Parkhill J, Hancock RE, Brinkman FS, Levesque RC (2009) Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool epidemic strain of Pseudomonas aeruginosa. Genome Res 19(1):12–23. https://doi.org/10.1101/gr.086082.108 CrossRefPubMedPubMedCentralGoogle Scholar
- Yan Q, Philmus B, Chang JH, Loper JE (2017) Novel mechanism of metabolic co-regulation coordinates the biosynthesis of secondary metabolites in Pseudomonas protegens. eLife 6:e22835. https://doi.org/10.7554/eLife.22835