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.

Preparation of Cyclic Prodiginines by Mutasynthesis in Pseudomonas putida KT2440

“…For the exploration of the mutasynthon acceptance in the mutasynthesis the cyclic 2,3‐alkylpyrroles 8 a – e were supplemented to cultures of the MBC‐producing strain P. putida pig‐r2‐Δ pigD (Scheme ). This engineered strain was derived from P. putida pig‐r2, a prodigiosin‐producing strain containing the Serratia marcescens pig gene cluster integrated into the chromosome . Prodigiosin production was inhibited by knocking out the gene encoding the first enzyme gene ( pigD ) of the monopyrrole 2‐methyl‐3‐amyl pyrrole ( 9 , MAP, C‐ring) biosynthesis.…”

“…Mutasynthesis (preparative scale) : Each reaction was performed in a Fernbach flask (3 L) with use of a final volume of 500 mL containing TB liquid medium, pyrrole precursor (0.5 m m ), and DMSO (2 %, v / v ). Polyurethane (PU) foam cubes (Bornewasser, Göllheim, Germany: Softpur, 25 kg m −3 density, 4 kPa compression hardness, 5 g), each approximately 1 cm 3 , were added as an adsorbent for the in situ extraction of prodiginines . In detail, the TB Sm liquid medium was inoculated with an overnight culture of P. putida pig‐r2 Δ pigD to an OD 600 of 0.02.…”

“…With regard to efficient accessibility of prodiginines, we have previously explored the heterologous production of prodigiosin ( 2 ) in the GRAS‐certified strain Pseudomonas putida KT2440 . Furthermore, we demonstrated the potential of a mutasynthesis approach to create more diversity in the C‐ring of this natural secondary metabolite .…”

Substrate Flexibility of the Flavin‐Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis

“…as pure prodigiosin has negligible absorbance at 450 and 570 nm, whereas extracts from strains not producing prodigiosin showed a fairly linear decrease in absorbance across this region, with the result that for a nonproducing strain was close to zero. Using a value for the extinction coefficient of prodigiosin of ϵ 535 =139 800 m −1 cm −1 , the average quantities of prodiginine produced from a 25 mL culture starting, from the H 2 MAP analogue shown, were 5 b , 11 nmol; 5 c , 55 nmol; 5 d , 34 nmol; 5 e , 72 nmol; 5 f , 22 nmol; 5 g , 1.2 nmol; 5 h , 21 nmol; 5 i , 4 nmol: 5 j and 5 k , 0 nmol. These experiments were run on four different occasions and on each occasion cultures fed with H 2 MAP 5 a were included as the positive control.…”

“…It is often achieved through the condensation of an analogue of monopyrrole 2‐methyl‐3‐amylpyrrole (MAP) 3 and the bipyrrole 4‐methoxy‐2,2′‐bipyrrole‐5‐carbaldehyde (MBC) 4 . MAP can be obtained in two steps from octan‐2‐one but, even though improvements have been made, formation of MBC and its analogues still requires complex multistep synthesis. A better understanding of the biosynthetic process leading to the formation of prodigiosin could help to address this challenge.…”