Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels.

Semi-rational redesign of the substrate binding pocket and access tunnels of prodigiosin ligase PigC enhanced the catalytic efficiency in the synthesis of pyrrolic anti-cancer agents more than 45 times. A molecular understanding was gained on residues V333 and T334 relevant to substrate binding and translocation of small pyrroles through PigC access tunnels.

New Prodigiosin Derivatives Obtained by Mutasynthesis in Pseudomonas putida.

The deeply red-colored natural compound prodigiosin is a representative of the prodiginine alkaloid family, which possesses bioactivities as antimicrobial, antitumor, and antimalarial agents. Various bacteria including the opportunistic human pathogen Serratia marcescens and different members of the Streptomycetaceae and Pseudoalteromonadaceae produce prodiginines. In addition, these microbes generally accumulate many structurally related alkaloids making efficient prodiginine synthesis and purification difficult and expensive. Furthermore, it is known that structurally different natural prodiginine variants display differential bioactivities. In the herein described mutasynthesis approach, 13 different derivatives of prodigiosin were obtained utilizing the GRAS (generally recognized as safe) classified strain Pseudomonas putida KT2440. Genetic engineering of the prodigiosin pathway together with incorporation of synthetic intermediates thus resulted in the formation of a so far unprecedented structural diversity of new prodiginine derivatives in P. putida. Furthermore, the formed products allow reliable conclusions regarding the substrate specificity of PigC, the final condensing enzyme in the prodigiosin biosynthesis pathway of S. marcescens. The biological activity of prodigiosin toward modulation of autophagy was preserved in prodiginine derivatives. One prodiginine derivative displayed more potent autophagy inhibitory activity than the parent compound or the synthetic clinical candidate obatoclax.

Efficient recombinant production of prodigiosin in Pseudomonas putida.

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.