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

Abstract

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 1 H and 13 C{ 1 H} NMR signals of caryoynencins were completely assigned by analyzing the NMR data of the isolated compounds and protegenin A enriched with [1-13 C]-or [2-13 C]-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.

Disproof of the Structures and Biosynthesis of Ergoynes, Gs-Polyyne–l-Ergothioneine Cycloadducts from Gynuella sunshinyii YC6258

“…To confirm the production of Gs-polyyne and ergoynes, we analyzed the culture extract of G. sunshinyii YC6258 by LC/MS, following our culturing and extraction methods for bacterial polyynes. 6,7,15 As a result, an LC peak with a UV spectrum characteristic for ene−tetrayne-type polyynes was detected (Figure 2A,B). 7,15 Its ESI-MS data were consistent with the estimated m/z value of Gs-polyyne [M + H] + (Figure 2B).…”

“…6,7,15 As a result, an LC peak with a UV spectrum characteristic for ene−tetrayne-type polyynes was detected (Figure 2A,B). 7,15 Its ESI-MS data were consistent with the estimated m/z value of Gs-polyyne [M + H] + (Figure 2B). Thus, we assumed that the detected compound was Gspolyyne (3).…”

“…Thereafter, the concentrated solution was generated by evaporating the EtOAc extract after the addition of N,N-dimethylformamide (DMF, 10 mL), followed by separation on an octadecylsilyl (ODS) column using an elution mixture (water and methanol (MeOH)). 7 As confirmed by LC/MS, Gs-polyyne (3) was detected in the 100% MeOH eluate. Reversed-phase HPLC combining ODS and cholesteryl columns effectively separated 3 from impurities.…”

“…Although the existence of bacterial polyynes has become well known, they are chemically unstable, requiring up-to-date attention regarding their extraction, isolation, and structural analysis. 6,7 Gynuella sunshinyii YC6258, a marine γ-proteobacterium, has been isolated from the rhizosphere of a halophyte (Carex scabrifolia Steud.). 8 This bacterial strain exhibits strong antifungal and antioomycete activities against a variety of plant pathogens.…”

“…We previously reported a method for performing the stable isolation and structural analysis of caryoynencins A−C bearing a diene−tetrayne moiety. 7,14 Caryoynencins exhibit the properties of polyynes and polyenes, which complicate their handling owing to their susceptibilities to polymerization and…”