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

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

1,* , 2
 and 2
Received: 9 December 2010; in revised form: 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.
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 which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

AMA Style

Youssef NH, Wofford N, McInerney MJ. Importance of the Long-Chain Fatty Acid Beta-Hydroxylating Cytochrome P450 Enzyme YbdT for Lipopeptide Biosynthesis in Bacillus subtilis Strain OKB105. International Journal of Molecular Sciences. 2011; 12(3):1767-1786.

Chicago/Turabian Style

Youssef, Noha H.; Wofford, Neil; McInerney, Michael J. 2011. "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. 12, no. 3: 1767-1786.



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