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Efficient recombinant production of prodigiosin in Pseudomonas putida.

ABSTRACT: Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

SUBMITTER: Domrose A

PROVIDER: S-EPMC4569968 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Efficient recombinant production of prodigiosin in Pseudomonas putida.

Domröse Andreas A Klein Andreas S AS Hage-Hülsmann Jennifer J Thies Stephan S Svensson Vera V Classen Thomas T Pietruszka Jörg J Jaeger Karl-Erich KE Drepper Thomas T Loeschcke Anita A

Frontiers in microbiology 20150915

Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas puti ...[more]

PMID: 26441905

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Project description:BackgroundBioethanol is one of the most representative eco-friendly fuels developed to replace the non-renewable fossil fuels and is the most successful commercially available bio-conversion technology till date. With the availability of inexpensive carbon sources, such as cellulosic biomass, bioethanol production has become cheaper and easier to perform, which can facilitate the development of methods for converting ethanol into higher value-added biochemicals. In this study, a bioconversion process using Pseudomonas putida as a biocatalyst was established, wherein ethanol was converted to mevalonate. Since ethanol can be converted directly to acetyl-CoA, bypassing its conversion to pyruvate, there is a possibility that ethanol can be converted to mevalonate without producing pyruvate-derived by-products. Furthermore, P. putida seems to be highly resistant to the toxicity caused by terpenoids, and thus can be useful in conducting terpenoid production research.ResultsIn this study, we first expressed the core genes responsible for mevalonate production (atoB, mvaS, and mvaE) in P. putida and mevalonate production was confirmed. Thereafter, through an improvement in genetic stability and ethanol metabolism manipulation, mevalonate production was enhanced up to 2.39-fold (1.70 g/L vs. 4.07 g/L) from 200 mM ethanol with an enhancement in reproducibility of mevalonate production. Following this, the metabolic characteristics related to ethanol catabolism and mevalonate production were revealed by manipulations to reduce fatty acid biosynthesis and optimize pH by batch fermentation. Finally, we reached a product yield of 0.41 g mevalonate/g ethanol in flask scale culture and 0.32 g mevalonate/g ethanol in batch fermentation. This is the highest experimental yield obtained from using carbon sources other than carbohydrates till date and it is expected that further improvements will be made through the development of fermentation methods.ConclusionPseudomonas putida was investigated as a biocatalyst that can efficiently convert ethanol to mevalonate, the major precursor for terpenoid production, and this research is expected to open new avenues for the production of terpenoids using microorganisms that have not yet reached the stage of mass production.

Project description:Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

Dataset Information

Efficient recombinant production of prodigiosin in Pseudomonas putida.

Publications

Efficient recombinant production of prodigiosin in Pseudomonas putida.

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