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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 generated by error prone PCR increase E. coli ΔfadR growth rate on octanoate without increasing FadD expression.(A) FadD catalyzes the first step in E. coli growth on fatty acids but has low activity on fatty acids shorter than 10 carbons. (B) Error prone PCR and FadD screening scheme (Materials and Methods). (C) Growth of E. coli ΔfadR expressing the indicated C-terminally His6-tagged FadD mutants generated by error prone PCR from vector pETDuet-1 on octanoate. (D) Relative increase in FadD expression (dark gray) and growth rate (light gray) conferred by His6-tagged FadD mutants on octanoate compared to wild-type FadD. n = 5 for FadD expression and 6 for growth rate; error bars indicate standard deviation. All increases in growth rate have p < 0.05 by two sided students T-test while all changes in expression have p > 0.3. FadD expression was measured using anti-his western blot samples normalized to total protein content by A280 (Materials and Methods).
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fig-1: FadD mutants generated by error prone PCR increase E. coli ΔfadR growth rate on octanoate without increasing FadD expression.(A) FadD catalyzes the first step in E. coli growth on fatty acids but has low activity on fatty acids shorter than 10 carbons. (B) Error prone PCR and FadD screening scheme (Materials and Methods). (C) Growth of E. coli ΔfadR expressing the indicated C-terminally His6-tagged FadD mutants generated by error prone PCR from vector pETDuet-1 on octanoate. (D) Relative increase in FadD expression (dark gray) and growth rate (light gray) conferred by His6-tagged FadD mutants on octanoate compared to wild-type FadD. n = 5 for FadD expression and 6 for growth rate; error bars indicate standard deviation. All increases in growth rate have p < 0.05 by two sided students T-test while all changes in expression have p > 0.3. FadD expression was measured using anti-his western blot samples normalized to total protein content by A280 (Materials and Methods).

Mentions: Medium chain fatty acids (MCFAs, 6–12 carbons) are important precursors to fuel-like compounds and industrial chemicals (Handke, Lynch & Gill, 2011; Knothe, 2009). E. coli have been engineered to produce MCFAs using a variety of techniques (Akhtar et al., 2015; Choi & Lee, 2013; Dehesh et al., 1996; Dellomonaco et al., 2011; Torella et al., 2013; Voelker & Davies, 1994), but their conversion into fuel-like compounds such as alcohols and alkanes requires activation of the MCFA carboxylic acid head group into a stronger electrophile. Biologically, this can be achieved by converting the carboxyl group into an acyl-CoA thioester. The acyl-CoA synthetase FadD catalyses this conversion in E. coli aerobic beta-oxidation and has been used to activate long chain fatty acids (LCFAs, 13 + carbons) for their later reduction into fuel-like compounds (Fig. 1) (Black et al., 1992; Doan et al., 2009; Steen et al., 2010; Zhang, Carothers & Keasling, 2012). However, FadD has low activity on fatty acids less than 10 carbons long resulting in slow E. coli growth rates on these fatty acids even in the presence of mutations de-repressing fadD and other genes involved in beta-oxidation (Campbell, Morgan-Kiss & Cronan, 2003; Iram & Cronan, 2006; Kameda & Nunn, 1981; Overath, Pauli & Schairer, 1969; Salanitro & Wegener, 1971). Salmonella enterica, which has a FadD very similar to that of E. coli, grows more quickly than E. coli on octanoate, but this is due to changes in fadD regulation and the activity of downstream beta-oxidation enzymes and not to changes in FadD enzymatic activity (Iram & Cronan, 2006).


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

Ford TJ, Way JC - PeerJ (2015)

FadD mutants generated by error prone PCR increase E. coli ΔfadR growth rate on octanoate without increasing FadD expression.(A) FadD catalyzes the first step in E. coli growth on fatty acids but has low activity on fatty acids shorter than 10 carbons. (B) Error prone PCR and FadD screening scheme (Materials and Methods). (C) Growth of E. coli ΔfadR expressing the indicated C-terminally His6-tagged FadD mutants generated by error prone PCR from vector pETDuet-1 on octanoate. (D) Relative increase in FadD expression (dark gray) and growth rate (light gray) conferred by His6-tagged FadD mutants on octanoate compared to wild-type FadD. n = 5 for FadD expression and 6 for growth rate; error bars indicate standard deviation. All increases in growth rate have p < 0.05 by two sided students T-test while all changes in expression have p > 0.3. FadD expression was measured using anti-his western blot samples normalized to total protein content by A280 (Materials and Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-1: FadD mutants generated by error prone PCR increase E. coli ΔfadR growth rate on octanoate without increasing FadD expression.(A) FadD catalyzes the first step in E. coli growth on fatty acids but has low activity on fatty acids shorter than 10 carbons. (B) Error prone PCR and FadD screening scheme (Materials and Methods). (C) Growth of E. coli ΔfadR expressing the indicated C-terminally His6-tagged FadD mutants generated by error prone PCR from vector pETDuet-1 on octanoate. (D) Relative increase in FadD expression (dark gray) and growth rate (light gray) conferred by His6-tagged FadD mutants on octanoate compared to wild-type FadD. n = 5 for FadD expression and 6 for growth rate; error bars indicate standard deviation. All increases in growth rate have p < 0.05 by two sided students T-test while all changes in expression have p > 0.3. FadD expression was measured using anti-his western blot samples normalized to total protein content by A280 (Materials and Methods).
Mentions: Medium chain fatty acids (MCFAs, 6–12 carbons) are important precursors to fuel-like compounds and industrial chemicals (Handke, Lynch & Gill, 2011; Knothe, 2009). E. coli have been engineered to produce MCFAs using a variety of techniques (Akhtar et al., 2015; Choi & Lee, 2013; Dehesh et al., 1996; Dellomonaco et al., 2011; Torella et al., 2013; Voelker & Davies, 1994), but their conversion into fuel-like compounds such as alcohols and alkanes requires activation of the MCFA carboxylic acid head group into a stronger electrophile. Biologically, this can be achieved by converting the carboxyl group into an acyl-CoA thioester. The acyl-CoA synthetase FadD catalyses this conversion in E. coli aerobic beta-oxidation and has been used to activate long chain fatty acids (LCFAs, 13 + carbons) for their later reduction into fuel-like compounds (Fig. 1) (Black et al., 1992; Doan et al., 2009; Steen et al., 2010; Zhang, Carothers & Keasling, 2012). However, FadD has low activity on fatty acids less than 10 carbons long resulting in slow E. coli growth rates on these fatty acids even in the presence of mutations de-repressing fadD and other genes involved in beta-oxidation (Campbell, Morgan-Kiss & Cronan, 2003; Iram & Cronan, 2006; Kameda & Nunn, 1981; Overath, Pauli & Schairer, 1969; Salanitro & Wegener, 1971). Salmonella enterica, which has a FadD very similar to that of E. coli, grows more quickly than E. coli on octanoate, but this is due to changes in fadD regulation and the activity of downstream beta-oxidation enzymes and not to changes in FadD enzymatic activity (Iram & Cronan, 2006).

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