Dataset Information

Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

ABSTRACT: Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

SUBMITTER: Jiang Y

PROVIDER: S-EPMC2888595 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

Jiang Yanfang Y Morgan-Kiss Rachael M RM Campbell John W JW Chan Chi Ho CH Cronan John E JE

Biochemistry 20100201 4

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty ac ...[more]

PMID: 20028080

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Project description:Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources into a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e. binding selectivity). Here, we demonstrate this approach by identifying medium-chain (C6-C12) acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, products of thioesterase-catalyzed hydrolysis, are limited in natural abundance compared to long-chain fatty acids; the limited supply leads to high costs of C6-C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding to the highly-active 'TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12- or C8-specificity while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified three thioesterases with enhanced production of dodecanoic acid (C12) and twenty-seven thioesterases with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of 'TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of 'TesA variants suggest that hydrophobic forces govern 'TesA substrate specificity. We expect that the design rules we uncovered and the thioesterase variants identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain oleochemicals.

Project description:The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M(r) 70,000) and FATP2b (M(r) 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14-C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.

Dataset Information

Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

Publications

Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

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