Thiotemplated Biosynthesis of Bacterial Polyyne Fatty Acids by a Designated Desaturase Triad

Abstract

Various bacterial species are capable of producing highly modified fatty acid derivatives with conjugated triple bonds, which play important ecological roles as antifungals and toxins in mutualistic and pathogenic interactions. Furthermore, the terminal polyyne moiety is of interest as pharmacophore and as tag in bioorthogonal chemistry and live imaging. To gain insight into the assembly of these highly reactive natural products, we investigated tetrayne (caryoynencin and protegencin) biosynthesis genes (cay and pgn) from Trinickia caryophylli and Pseudomonas protegens. Pathway dissection and reconstitution in the heterologous host Burkholderia graminis revealed the genes minimally required for polyyne formation. Mutational analyses and biochemical assays demonstrated that polyyne biosynthesis is thiotemplated, involving a fatty acyl‐AMP ligase, a designated acyl carrier protein, and a thioesterase. Heterologous expression of point‐mutated desaturase genes showed that three desaturases work synergistically to introduce four triple bonds. These findings point to an intricate desaturase complex and provide important information for future bioengineering experiments.

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

“…Recent articles have described a possible pathway for protegenin/caryoynencin biosynthesis in which the precursor fatty acid is elaidic acid. , However, to the best of our knowledge, there are no supporting experimental data. To identify a precursor, feeding experiments of candidate C 18 fatty acids (stearic, elaidic, and oleic acids) with P.…”

“…First, ProA (fatty acyl-AMP ligase) activates the precursor (stearic acid), loading it into ProD (ACP) and yielding a stearyl-ACP conjugate. , Thereafter, ProB (desaturase/acetylenase), ProC (desaturase/acetylenase), and ProE (desaturase domain) successively act on stearyl-ACP to construct the ene–tetrayne moiety. , ProF may be involved in electron transfer during the ene–tetrayne formation . The thioesterase activity of ProE causes the fatty acid part to be cleaved from the ACP complex, thereby yielding protegenin A ( 6 ). ,, From the amino acid sequence, ProG (α/β-hydrolase) was expected to play a role in assisting the thioesterase activity of ProE. The application of CayG to protegenin A ( 6 ) caused a series of hydroxylation, dehydration, and hydroxylation reactions to afford caryoynencins A–C ( 1 – 3 ).…”

“…25,26 Thereafter, ProB (desaturase/acetylenase), ProC (desaturase/acetylenase), and ProE (desaturase domain) successively act on stearyl-ACP to construct the ene−tetrayne moiety. 23,24 ProF may be involved in electron transfer during the ene−tetrayne formation. 11 The thioesterase activity of ProE causes the fatty acid part to be cleaved from the ACP complex, thereby yielding protegenin A (6).…”

“…11 The thioesterase activity of ProE causes the fatty acid part to be cleaved from the ACP complex, thereby yielding protegenin A (6). 11,23,24 From the amino acid sequence, ProG (α/β-hydrolase) was expected to play a role in assisting the thioesterase activity of ProE. The application of CayG to protegenin A (6) caused a series of hydroxylation, dehydration, and hydroxylation reactions to afford caryoynencins A−C (1−3).…”

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

“…The comparison of the pol and pro BGCs revealed that the encoded enzymes and proteins were the same, except for proGH (Figure C). The carbon skeleton of bacterial polyynes is determined by the type of fatty acid loaded to the corresponding ACP by fatty acyl-adenosine monophosphate (AMP) ligase. − Thus, we generated the phylogenetic tree of fatty acyl-AMP ligases from the bacterial polyyne BGCs registered in the GenBank database using a maximum likelihood method (Figure C). , The result revealed a clear division between C 16 ( upper ) and C 18 type ( lower ) parts of the phylogenetic tree. The C 16 subtree contains the clades of Streptomyces polyynes ( green ), cepacins ( purple ), and collimonins ( orange ), , whereas the C 18 subtree contains the clades of protegenins ( red ) , and caryoynencins ( blue ). , PolA from G.…”