DISCOVERY AND INVESTIGATION OF THE BIOSYNTHESIS OF A NOVEL LANTHIPEPTIDE MOLECULE FROM A MARINE BACTERIUM Pseudoalteromonas flavipulchra 2ta6

Abstract

Bacterial natural products have been a rich source of therapeutics since ancient times. Genome sequencing has shown the potential of bacteria to produce even more Natural Product molecules. Lanthipeptides are ribosomally synthesized and post-translationally modified peptide (RiPP) molecules characterized by the presence of thioether crosslinks known as lanthionines. Lanthipeptides are classified into four classes based on the biosynthetic enzymes involved to install the thioether crosslinks. In class I lanthipeptides, two distinct enzymes, a dehydratase and a cyclase are employed for this purpose whereas in other classes the thioether crosslinks are formed by a bifunctional enzyme. Development of genome mining tools have made it possible to find lanthipeptide encoding gene clusters from the bacteria of multiple different phyla. Lanthipeptide structures can be predicted based on gene sequence information and the types of post-translational modification enzymes present, enabling the isolation and chemical structure elucidation of these RiPPs allowing subsequent bioactivity testing and investigation of the biosynthesis. This thesis describes the isolation the new lanthipeptide molecule flavipuricin I (18) from a gram-negative marine proteobacteria Pseudoalteromonas flavipulchra 2ta6 and the attempts taken for in vitro and in vivo production using Escherichia coli as a heterologous host. A genome mining approach combined with mass spectrometry guided isolation and structure elucidation was adopted for discovering flavipuricin I (18). Also, the co-expression of genes for synthetases from flavipuricin I (18) biosynthetic pathway, yielded post-translationally modified flavipuricin I (18). The biosynthetic enzymes were successfully purified for a future in vitro biosynthesis of flavipuricin I (18) . Further, the identification of a peptidase containing transporter (FlnT) suggests a new role for bifunctional transporters in class I lanthipeptide biosynthesis. To the best of our knowledge flavipuricin I (18) is the first lanthipeptide isolated from a proteobacterium, suggesting this phylum may be a promising future source of new lanthipeptides