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Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.

Ford TJ, Way JC - PeerJ (2015)

Bottom Line: This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes.Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit.These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School , Boston, MA , USA.

ABSTRACT
FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6-12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

No MeSH data available.


Related in: MedlinePlus

FadD mutants enhance the growth rate of E.coli ΔfadR on the MCFAs hexanoate, octanoate, and decanoate, but not on palmitate and oleate.E.coli ΔfadR transformed with empty pETDuet-1 (black) C-terminally His6-tagged wild-type fadD (blue) or the indicated C-terminally His6-tagged fadD mutants (Red) were grown on minimal medium containing the indicated fatty acid as the sole carbon source. Growth rates were measured by linear regression of the normalized log2(OD590) during exponential phase. n = 3, errors bars indicate standard deviation, and ** indicates p < 0.05 compared to wild-type by two sided students T-test.
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fig-2: FadD mutants enhance the growth rate of E.coli ΔfadR on the MCFAs hexanoate, octanoate, and decanoate, but not on palmitate and oleate.E.coli ΔfadR transformed with empty pETDuet-1 (black) C-terminally His6-tagged wild-type fadD (blue) or the indicated C-terminally His6-tagged fadD mutants (Red) were grown on minimal medium containing the indicated fatty acid as the sole carbon source. Growth rates were measured by linear regression of the normalized log2(OD590) during exponential phase. n = 3, errors bars indicate standard deviation, and ** indicates p < 0.05 compared to wild-type by two sided students T-test.

Mentions: The FadD mutants generated by error-prone PCR do not increase FadD expression. To ensure that the FadD mutants do not increase growth rate by simply enhancing FadD expression, wild-type FadD and the FadD mutants were C-terminally His6-tagged, growth was measured (Fig. 1B), and SDS-PAGE samples were prepared at early exponential phase (∼26 h of growth in octanoate minimal medium). Samples were normalized for total protein by A280 and western blotted with an anti-his antibody (Materials and Methods). While increases in growth rate were very consistent (p < 0.05 in all cases), there was no significant difference in FadD expression between the wild-type and mutant variants (Fig. 2C).


Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids.

Ford TJ, Way JC - PeerJ (2015)

FadD mutants enhance the growth rate of E.coli ΔfadR on the MCFAs hexanoate, octanoate, and decanoate, but not on palmitate and oleate.E.coli ΔfadR transformed with empty pETDuet-1 (black) C-terminally His6-tagged wild-type fadD (blue) or the indicated C-terminally His6-tagged fadD mutants (Red) were grown on minimal medium containing the indicated fatty acid as the sole carbon source. Growth rates were measured by linear regression of the normalized log2(OD590) during exponential phase. n = 3, errors bars indicate standard deviation, and ** indicates p < 0.05 compared to wild-type by two sided students T-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493641&req=5

fig-2: FadD mutants enhance the growth rate of E.coli ΔfadR on the MCFAs hexanoate, octanoate, and decanoate, but not on palmitate and oleate.E.coli ΔfadR transformed with empty pETDuet-1 (black) C-terminally His6-tagged wild-type fadD (blue) or the indicated C-terminally His6-tagged fadD mutants (Red) were grown on minimal medium containing the indicated fatty acid as the sole carbon source. Growth rates were measured by linear regression of the normalized log2(OD590) during exponential phase. n = 3, errors bars indicate standard deviation, and ** indicates p < 0.05 compared to wild-type by two sided students T-test.
Mentions: The FadD mutants generated by error-prone PCR do not increase FadD expression. To ensure that the FadD mutants do not increase growth rate by simply enhancing FadD expression, wild-type FadD and the FadD mutants were C-terminally His6-tagged, growth was measured (Fig. 1B), and SDS-PAGE samples were prepared at early exponential phase (∼26 h of growth in octanoate minimal medium). Samples were normalized for total protein by A280 and western blotted with an anti-his antibody (Materials and Methods). While increases in growth rate were very consistent (p < 0.05 in all cases), there was no significant difference in FadD expression between the wild-type and mutant variants (Fig. 2C).

Bottom Line: This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes.Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit.These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School , Boston, MA , USA.

ABSTRACT
FadD catalyses the first step in E. coli beta-oxidation, the activation of free fatty acids into acyl-CoA thioesters. This activation makes fatty acids competent for catabolism and reduction into derivatives like alcohols and alkanes. Alcohols and alkanes derived from medium chain fatty acids (MCFAs, 6-12 carbons) are potential biofuels; however, FadD has low activity on MCFAs. Herein, we generate mutations in fadD that enhance its acyl-CoA synthetase activity on MCFAs. Homology modeling reveals that these mutations cluster on a face of FadD from which the co-product, AMP, is expected to exit. Using FadD homology models, we design additional FadD mutations that enhance E. coli growth rate on octanoate and provide evidence for a model wherein FadD activity on octanoate can be enhanced by aiding product exit. These studies provide FadD mutants useful for producing MCFA derivatives and a rationale to alter the substrate specificity of adenylating enzymes.

No MeSH data available.


Related in: MedlinePlus