Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase.

Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.

Bioinformatic, phylogenetic and chemical analysis of the UV-absorbing compounds scytonemin and mycosporine-like amino acids from the microbial mat communities of Shark Bay, Australia.

Shark Bay, Western Australia is a World Heritage area with extensive microbial mats and stromatolites. Microbial communities that comprise these mats have developed a range of mitigation strategies against changing levels of photosynthetically active and ultraviolet radiation, including the ability to biosynthesise the UV-absorbing natural products scytonemin and mycosporine-like amino acids (MAAs). To this end, the distribution of photoprotective pigments within Shark Bay microbial mats was delineated in the present study. This involved amplicon sequencing of bacterial 16S rDNA from communities at the surface and subsurface in three distinct mat types (smooth, pustular and tufted), and correlating this data with the chemical and molecular distribution of scytonemin and MAAs. Employing UV spectroscopy and MS/MS fragmentation, mycosporine-glycine, asterina and an unknown MAA were identified based on typical fragmentation patterns. Marker genes for scytonemin and MAA production (scyC and mysC) were amplified from microbial mat DNA and placed into phylogenetic context against a broad screen throughout 363 cyanobacterial genomes. Results indicate that occurrence of UV screening compounds is associated with the upper layer of Shark Bay microbial mats, and the occurrence of scytonemin is closely dependent on the abundance of cyanobacteria.