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Int. J. Mol. Sci. 2011, 12(3), 1767-1786; doi:10.3390/ijms12031767

Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105

1
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA
2
Department of Botany and Microbiology, University of Oklahoma, Norman, OK 74078, USA
*
Author to whom correspondence should be addressed.
Received: 9 December 2010 / Revised: 8 February 2011 / Accepted: 3 March 2011 / Published: 8 March 2011
(This article belongs to the Special Issue Biosurfactants)
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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 H2O2, 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. View Full-Text
Keywords: cytochrome P450; YbdT; long chain fatty acids; beta hydroxylation; Bacillus subtilis; surfactin cytochrome P450; YbdT; long chain fatty acids; beta hydroxylation; Bacillus subtilis; surfactin
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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MDPI and ACS Style

Youssef, N.H.; Wofford, N.; McInerney, M.J. Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105. Int. J. Mol. Sci. 2011, 12, 1767-1786.

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