A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line

Abstract

The antimicrobial secondary metabolite kalimantacin (also called batumin) is produced by a hybrid polyketide/non-ribosomal peptide system in Pseudomonas fluorescens BCCM_ID9359. In this study, the kalimantacin biosynthesis gene cluster is analyzed by yeast two-hybrid analysis, creating a protein–protein interaction map of the entire assembly line. In total, 28 potential interactions were identified, of which 13 could be confirmed further. These interactions include the dimerization of ketosynthase domains, a link between assembly line modules 9 and 10, and a specific interaction between the trans-acting enoyl reductase BatK and the carrier proteins of modules 8 and 10. These interactions reveal fundamental insight into the biosynthesis of secondary metabolites. This study is the first to reveal interactions in a complete biosynthetic pathway. Similar future studies could build a strong basis for engineering strategies in such clusters.

Control of β‐Branching in Kalimantacin Biosynthesis: Application of 13C NMR to Polyketide Programming

“…Two saturated β‐methyl branches are also formed in kalimantacin biosynthesis, first, when the biosynthetic intermediate is attached to the ACP in module 6 located on Bat2, and second, when attached to ACP5 on Bat3 (module 12). The saturated β‐methyl is hypothesised to be formed through the reduction of an HCS cassette derived endo ‐β‐methyl by a trans‐acting ER domain (BatK) . To elucidate the selectivity of BatK, ACPs derivatised with 6 were incubated with BatK and NADH and assayed by MS (Figure ) …”

Control of β‐Branching in Kalimantacin Biosynthesis: Application of 13C NMR to Polyketide Programming

“…The saturated bmethyl is hypothesised to be formed via the reduction of an HCS cassette derived endo-b-methyl by a trans-acting ER domain (BatK). [1,14] To elucidate the selectivity of BatK, ACPs derivatised with 6 were incubated with BatK and NADH and assayed by MS (Figure 5). [15] The MS-eject assay with ACP5 showed the production of a new ion (345 Da) after 1 h and complete consumption of the starting material (343 Da) confirming reduction as expected.…”