Structures and Biosynthesis of Caryoynencins, Unstable Bacterial Polyynes from Pseudomonas protegens Recombinant Expressing the cayG Gene.

Bacteria in certain genera can produce "bacterial polyynes" that contain a conjugated C≡C bond starting from a terminal alkyne. Protegenin A is a derivative of octadecanoic acid that contains an ene-tetrayne moiety. It was discovered in Pseudomonas protegens Cab57 and exhibits strong antioomycete and moderate antifungal activity. By introducing cayG, a cytochrome P450 gene from Burkholderia caryophylli, into P. protegens Cab57, protegenin A was converted into more complex polyynes, caryoynencins A-E. A purification method that minimized the degradation and isomerization of caryoynencins was established. For the first time, as far as we know, the 1H and 13C{1H} NMR signals of caryoynencins were completely assigned by analyzing the NMR data of the isolated compounds and protegenin A enriched with [1-13C]- or [2-13C]-acetate. Through the structural analysis of caryoynencins D/E and bioconversion experiments, we observed that CayG constructs the allyl alcohol moiety of caryoynencins A-C through sequential hydroxylation, dehydration, and hydroxylation. The recombinant strain exhibited a stronger antioomycete activity compared to the wild-type strain. This paper proposes a stable purification and structural determination method for various bacterial polyynes, and P. protegens Cab57 holds promise as an engineering host for the production of biologically active polyynes.

Genome Mining Discovery of Protegenins A-D, Bacterial Polyynes Involved in the Antioomycete and Biocontrol Activities of Pseudomonas protegens.

Some bacteria uniquely produce "bacterial polyynes", which possess a conjugated C≡C bond starting with a terminal alkyne, and use them as chemical weapons against hosts and competitors. Pseudomonas protegens Cab57, a biocontrol agent against plant pathogens, has an orphan biosynthetic gene cluster for bacterial polyynes (named protegenins). In this study, the isolation, structure elucidation, and biological characterization of protegenins A-D are reported. The structures of protegenins A-D determined by spectroscopic and chemical techniques were octadecanoic acid derivatives possessing an ene-tetrayne, ene-triyne-ene, or ene-triyne moiety. The protegenins exhibited weak to strong antioomycete activity against Pythium ultimum OPU774. The deletion of proA, a protegenin biosynthetic gene, resulted in the reduction of the antioomycete activity of P. protegens. The Gac/Rsm system, a quorum sensing-like system of Pseudomonas bacteria, regulated the production of protegenins. The production profile of protegenins was dependent on the culturing conditions, suggesting a control mechanism for protegenin production selectivity. P. protegens suppressed the damping-off of cucumber seedlings caused by P. ultimum, and this protective effect was reduced in the proA-deletion mutant. Altogether, protegenins are a new class of bacterial polyynes which contribute to the antioomycete and plant-protective effects of P. protegens.