Engineering Escherichia coli to synthesize free fatty acids

Lennen, Rebecca M.; Pfleger, Brian F.

doi:10.1016/j.tibtech.2012.09.006

Cited by 175 publications

(167 citation statements)

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“…The deletion of fadD, encoding an acyl-CoA synthetase, has been utilized in numerous studies to facilitate the accumulation of longer-chain carboxylic acids by preventing the uptake/activation of free fatty acids (14). Under these conditions, the deletion of fadD in JC01(DE3) atoB CT5 fadBA CT5 with pETDuet-fabI resulted in the extracellular production of 19.4 Ϯ 0.1 mg/liter tetradecanoic acid (C 14 ) and a slight increase in dodecanoic acid (C 12 ), with minimal impact on the levels of C 4 to C 10 carboxylic acids produced (Fig.…”

Section: Ct5mentioning

confidence: 99%

“…Among the aforementioned pathways, the bacterial type II fatty acid biosynthesis system (FAB II) (13) is probably the most widely engineered and has been harnessed for the synthesis of many products, including fatty acids (14), fatty acid methyl esters (15), fatty acid ethyl esters (11,16), fatty alcohols (11), and alkanes (12). At the core of the FAB II system is an elongation cycle that uses discrete enzymes to catalyze each of its four steps.…”

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confidence: 99%

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Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Vick

Clomburg

Blankschien

et al. 2015

Appl Environ Microbiol

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We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the ␤-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541-554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a ␤-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ⌬fabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the ␤-oxidation reversal in E. coli. R ecent advances in synthetic biology and enzyme and metabolic engineering (1, 2) are allowing the development of an ever-expanding array of microbial hosts for the production of advanced fuels and industrially relevant chemicals (3, 4). Most efforts are now focused on moving past the historically successful biofuels and industrial products, such as ethanol (5), toward "drop-in" fuels and additives that can be incorporated into existing infrastructure (4). The production of such molecules requires the synthesis of higher-chain-length (C Ն 4) products from 1-, 2-or 3-carbon metabolic intermediates and hence pathways that can mediate the formation of carbon-carbon bonds. A variety of native and engineered pathways have been utilized for this purpose, including the clostridial n-butanol pathway (6), isoprenoid biosynthesis (7,8), the ␣-keto-acids pathway (9, 10), and fatty acid biosynthesis (11,12).Among the aforementioned pathways, the bacterial type II fatty acid biosynthesis system (FAB II) (13) is probably the most widely engineered and has been harnessed for the synthesis of many products, including fatty acids (14), fatty acid methyl esters (15), fatty acid ethyl esters (11, 16), fatty alcohols (11), and alkanes (12). At the core of the FAB II system is an elongation cycle that uses discrete enzymes to catalyze each of its four ste...

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Section: Ct5mentioning

confidence: 99%

mentioning

confidence: 99%

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Vick

Clomburg

Blankschien

et al. 2015

Appl Environ Microbiol

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“…In E coli, thioesterases cleave acyl-ACPs producing the free fatty acids necessary for regulatory signals, export or degradation (Lennen and Pfleger, 2012). The fatty acid composition of E. coli expressing Camelina acyl-ACP thioesterase genes were analyzed and compared with control cells transformed with the empty pQE-80L vector (Table 3).…”

Section: Fatty Acid Analysis Of E Coli Expressing Camelina Sativa Acmentioning

confidence: 99%

“…These results showed that C. sativa thioesterases alter E. coli fatty acid metabolism, diverting the acyl chains away from the fatty acid and lipid biosynthetic pathways. These free fatty acids would later be secreted or degraded in the β-oxidation pathway (Lennen and Pfleger, 2012). The main change in the E. coli fatty acid composition when CsFatA was expressed was the reduction in unsaturated fatty acids, mainly oleic acid (18:1ω9).…”

Section: Fatty Acid Analysis Of E Coli Expressing Camelina Sativa Acmentioning

confidence: 99%

Acyl-ACP thioesterases from Camelina sativa: Cloning, enzymatic characterization and implication in seed oil fatty acid composition

et al. 2014

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Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into glycerolipids. Acyl-ACP thioesterases are classified into two families, FatA and FatB, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. Camelina sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of Camelina sativa oil was discussed in the light of these results.

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“…Fatty acids can be reduced to aldehydes and can then be subjected to decarbonylation, sequential reduction, or decarboxylation to produce equivalent alkanes as petroleum distillates ( Fig. 3) (Lennen and Pfleger 2012). Fatty acid biosynthesis is one of the anabolic pathways capable of producing large hydrophobic fatty acid molecules and their derivatives (Fig.…”

Section: Fats Lipids and Estersmentioning

confidence: 99%

Can Microbially Derived Advanced Biofuels Ever Compete with Conventional Bioethanol? A Critical Review

Veettil

Kumar

Koukoulas³

2016

BioResources

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Engineering Escherichia coli to synthesize free fatty acids

Cited by 175 publications

References 78 publications

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Acyl-ACP thioesterases from Camelina sativa: Cloning, enzymatic characterization and implication in seed oil fatty acid composition

Can Microbially Derived Advanced Biofuels Ever Compete with Conventional Bioethanol? A Critical Review

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