Characterization of Collimonin Biosynthesis in the Fungus-Feeding Bacterium Collimonas fungivorans Ter331

Collimonins A-D are bacterial polyynes produced by the fungus-feeding bacterium Collimonas fungivorans Ter331. The col biosynthetic gene cluster (BGC) is implicated in collimonin biosynthesis, but their gene functions remain unclear. In this study, the biosynthetic pathway of collimonins in C. fungivorans Ter331 was elucidated through genetic and chemical characterization of each col gene's function. The biosynthetic intermediates (namely, CBIs-1-4) were isolated from gene-deletion mutants (or with gene overexpression), and their structures were elucidated by spectroscopic analyses. The conversion of these intermediates to other intermediates or collimonins was analyzed by mutant coculturing, gene overexpression, and feeding experiments. CBI-1, which is the first detectable intermediate, was converted into collimonins A-D through desaturation, oxidation, and lactonization. Results indicated that the Rieske oxygenase ColK, the fatty acid desaturase ColJ, the possible cyclase ColM, and the flavin-dependent monooxygenase ColI were involved in the conversion of CBI-1 to collimonins A and B, whereas only ColK was responsible for its conversion to collimonins C and D. The mas BGC of Massilia armeniaca ZMN-3, which has a similar gene set to the col BGC but lacks a homologue of colI, can produce collimonins A-D when coexpressed with colI. A comparison of the antifungal activity between Ter331 and its col mutants suggested the importance of the latter lactonization in collimonin biosynthesis for the activity. This study represents the first comprehensive description of the biosynthetic pathway for collimonins and establishes an important basis for future biochemical analyses of each conversion step.