Biochemical Studies on the Biosynthetic Pathway of the Lantibiotic Microbisporicin

Lantibiotics are ribosomally synthesized antimicrobial peptides that undergo posttranslational modifications. Microbisporicin is one of the most potent and heavily modified lantibiotic, having 58% of its amino acids post-translationally modified. Among these modifications, microbisporicin contains two novel ones not found in any other known lantibiotic: the presence of 5-chlorotryptophan and 4,3-dihydroxyproline residues. BLAST analysis of the proteins within the biosynthetic gene cluster revealed the presence of a putative halogenase (MibH) and a P450 monooxygenase (MibO). Previous studies confirmed MibH function as tryptophan-halogenase. However, the enzymatic mechanism, substrate promiscuity, and the role of these modifications in its antimicrobial activity remain unclear. To understand the biosynthesis and biological function of these modifications, we are using in vivo and in vitro methods. For in vivo, a heterologous co-expression system is employed where the enzymes carrying out the post-translational modifications are co-expressed with the precursor peptide. The co-expression system serves to produce dehydrated, cyclized and C-terminal decarboxylated microbisporicin in vivo. This substrate is then used in in vitro enzymatic studies of MibH and MibO to elucidate their substrate requirements. We hypothesize that these proteins serve as tailoring enzymes acting at the later steps of microbisporicin biosynthesis. As such they will be active on the dehydrated-cyclized peptide. Using the co-expression system we were able to obtain C-terminal decarboxylated and 3-fold dehydrated microbisporicin. Further efforts include optimizing the expression system and performing kinetic studies on MibH and MibO. Such understanding will enable us to exploit the enzyme’s biotechnological potential of creating novel lantibiotic analogs carrying these modifications.