Expression of Secondary Metabolite Gene Clusters and Production of Secondary Metabolites in Three Nostoc strains Subjected to Deprivation on Nutrients and Competition

Abstract

Cyanobacteria are a unique source of natural products, where most of them are synthesized by non-ribosomal peptide synthase, polyketide synthase, or a hybrid of these pathways. The identification of such biosynthetic genes responsible for the production of secondary metabolites is still a relatively unexplored area, and it remains many natural products for which a biosynthetic origin is unknown. Secondary metabolites from marine cyanobacteria have gained much attention the last decades, however few comprehensive studies on secondary metabolites and their biosynthetic gene clusters from terrestrial cyanobacteria have been conducted. Three terrestrial Nostoc spp. KVJ20, KVJ2, and KVJ10 were recently sequenced which allowed us to conduct genome-wide predictions by AntiSMASH of their potential to produce secondary metabolites. These strains were subjected to various cultivation conditions including nutrient limitations and competition. Gene expression analysis by RT-qPCR of predicted gene clusters and the production of secondary metabolites by UPLC-HR-MS were conducted. Analysis of gene expression patterns revealed higher expression of several NRPS, PKS and RiPP genes in nutrient-deprived media, as well as confirming the present known function of the housekeeping genes; NifH, GvpC, and PilT. Most of the secondary metabolites found by UPLC-HR-MS were not identified, however a variety of Nostocyclopeptides, Suomilide/Banyaside-like peptides, Anabaenopeptins, as well as Aeruginosin, Hapalosin, and Nosperin were recorded from extracts of the respective strains. The results from this thesis give valuable knowledge for further cultivation of terrestrial cyanobacteria, with the purpose of awaking cryptic gene clusters and identifying novel secondary metabolites. We have also suggested conditions most suitable for enrichment for identified compounds.