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Importance of the long-chain fatty acid beta-hydroxylating cytochrome P450 enzyme YbdT for lipopeptide biosynthesis in Bacillus subtilis strain OKB105.

Youssef NH, Wofford N, McInerney MJ - Int J Mol Sci (2011)

Bottom Line: 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.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.Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.

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.

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β-hemolytic activity of the OKB105 cells compared to NHY1, the ybdT mutant. Blood agar plate was streaked with a single colony of OKB105 (wild-type) or NHY1 (ybdT mutant) cells. β-Hemolysis occurred within 24 h in OKB105 cells with clearing of blood agar and the appearance of a green sheen. NHY1 cells showed delayed hemolysis, which occurred after 48 h of incubation, and was not as extensive as that observed with OKB105 cells. The picture was taken 24 h after streaking.
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f1-ijms-12-01767: β-hemolytic activity of the OKB105 cells compared to NHY1, the ybdT mutant. Blood agar plate was streaked with a single colony of OKB105 (wild-type) or NHY1 (ybdT mutant) cells. β-Hemolysis occurred within 24 h in OKB105 cells with clearing of blood agar and the appearance of a green sheen. NHY1 cells showed delayed hemolysis, which occurred after 48 h of incubation, and was not as extensive as that observed with OKB105 cells. The picture was taken 24 h after streaking.

Mentions: The initiation of surfactin synthesis involves the transfer of the 3-hydroxyacyl moiety from 3-hydroxyacyl-CoA to SrfA [22]. Whether the 3-hydroxyacyl moiety of surfactin is derived from 3-hydroxyacyl-CoA generated in the beta-oxidation pathway or from the 3-hydroxylation of LCFAs is not known. We hypothesized that the LCFA 3-hydroxylating enzyme, YbdT, is responsible for the 3-hydroxylation of the fatty acids used for surfactin synthesis. To test this hypothesis, we mutated the ybdT via transformation of competent B. subtilis OKB105 cells with a PCR construct carrying a chloramphenicol-resistance cassette in-frame with the ybdT gene sequence. The competence of OKB105 was checked with a PCR product of rpoB gene that confers resistance to rifampicin. The percent transformation of OKB105 cells with rpoB gene was 2.05 × 10−4, comparable to a percent transformation of 7.7 × 10−4 reported previously [41]. A chloramphenicol-resistant mutant with delayed hemolysis of blood agar (NHY1) was obtained as a potential ybdT− mutant (Figure 1). Amplification and sequencing of the ybdT gene from NHY1 showed that the chloramphenicol-resistance cassette was inserted into the ybdT gene, confirming that NHY1 was a ybdT mutant.


Importance of the long-chain fatty acid beta-hydroxylating cytochrome P450 enzyme YbdT for lipopeptide biosynthesis in Bacillus subtilis strain OKB105.

Youssef NH, Wofford N, McInerney MJ - Int J Mol Sci (2011)

β-hemolytic activity of the OKB105 cells compared to NHY1, the ybdT mutant. Blood agar plate was streaked with a single colony of OKB105 (wild-type) or NHY1 (ybdT mutant) cells. β-Hemolysis occurred within 24 h in OKB105 cells with clearing of blood agar and the appearance of a green sheen. NHY1 cells showed delayed hemolysis, which occurred after 48 h of incubation, and was not as extensive as that observed with OKB105 cells. The picture was taken 24 h after streaking.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3111633&req=5

f1-ijms-12-01767: β-hemolytic activity of the OKB105 cells compared to NHY1, the ybdT mutant. Blood agar plate was streaked with a single colony of OKB105 (wild-type) or NHY1 (ybdT mutant) cells. β-Hemolysis occurred within 24 h in OKB105 cells with clearing of blood agar and the appearance of a green sheen. NHY1 cells showed delayed hemolysis, which occurred after 48 h of incubation, and was not as extensive as that observed with OKB105 cells. The picture was taken 24 h after streaking.
Mentions: The initiation of surfactin synthesis involves the transfer of the 3-hydroxyacyl moiety from 3-hydroxyacyl-CoA to SrfA [22]. Whether the 3-hydroxyacyl moiety of surfactin is derived from 3-hydroxyacyl-CoA generated in the beta-oxidation pathway or from the 3-hydroxylation of LCFAs is not known. We hypothesized that the LCFA 3-hydroxylating enzyme, YbdT, is responsible for the 3-hydroxylation of the fatty acids used for surfactin synthesis. To test this hypothesis, we mutated the ybdT via transformation of competent B. subtilis OKB105 cells with a PCR construct carrying a chloramphenicol-resistance cassette in-frame with the ybdT gene sequence. The competence of OKB105 was checked with a PCR product of rpoB gene that confers resistance to rifampicin. The percent transformation of OKB105 cells with rpoB gene was 2.05 × 10−4, comparable to a percent transformation of 7.7 × 10−4 reported previously [41]. A chloramphenicol-resistant mutant with delayed hemolysis of blood agar (NHY1) was obtained as a potential ybdT− mutant (Figure 1). Amplification and sequencing of the ybdT gene from NHY1 showed that the chloramphenicol-resistance cassette was inserted into the ybdT gene, confirming that NHY1 was a ybdT mutant.

Bottom Line: 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.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.Manipulation of LCFA specificity of YbdT could provide a new route to produce biosurfactants with activities tailored to specific functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.

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.

Show MeSH
Related in: MedlinePlus