title: Importance of the long-chain fatty acid beta-hydroxylating cytochrome P450 enzyme YbdT for lipopeptide biosynthesis in Bacillus subtilis strain OKB105.
abstract: Bacillus species produce extracellular, surface-active lipopeptides such as surfactin that have wide applications in industry and medicine. The steps involved in the synthesis of 3-hydroxyacyl-coenzyme A (CoA) substrates needed for surfactin biosynthesis are not understood. Cell-free extracts of Bacillus subtilis strain OKB105 synthesized lipopeptide biosurfactants in presence of l-amino acids, myristic acid, coenzyme A, ATP, and H(2)O(2), which suggested that 3-hydroxylation occurs prior to CoA ligation of the long chain fatty acids (LCFAs). We hypothesized that YbdT, a cytochrome P450 enzyme known to beta-hydroxylate LCFAs, functions to form 3-hydroxy fatty acids for lipopeptide biosynthesis. An in-frame mutation of ybdT was constructed and the resulting mutant strain (NHY1) produced predominantly non-hydroxylated lipopeptide with diminished biosurfactant and beta-hemolytic activities. Mass spectrometry showed that 95.6% of the fatty acids in the NHY1 biosurfactant were non-hydroxylated compared to only ∼61% in the OKB105 biosurfactant. Cell-free extracts of the NHY1 synthesized surfactin containing 3-hydroxymyristic acid from 3-hydroxymyristoyl-CoA at a specific activity similar to that of the wild type (17 ± 2 versus 17.4 ± 6 ng biosurfactant min(-1)·ng·protein(-1), respectively). These results showed that the mutation did not affect any function needed to synthesize surfactin once the 3-hydroxyacyl-CoA substrate was formed and that YbdT functions to supply 3-hydroxy fatty acid for surfactin biosynthesis. The fact that YbdT is a peroxidase could explain why biosurfactant production is rarely observed in anaerobically grown Bacillus species. Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions.
authors.affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Noha H
authors.initials: NH
authors.lastname: Youssef
authors.forename: Neil
authors.initials: N
authors.lastname: Wofford
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McInerney
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D055666:Lipopeptides / Q000096:biosynthesis* / Q000737:chemistry; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009227:Myristic Acids / Q000737:chemistry / Q000378:metabolism; D010456:Peptides, Cyclic / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D055666:Lipopeptides; D009227:Myristic Acids; D010456:Peptides, Cyclic; C009893:beta-hydroxymyristic acid; C009365:surfactin peptide; D003577:Cytochrome P-450 Enzyme System