Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle
| Autor: | Ramon Gonzalez, Alexander Chou, Seohyoung Kim, Matthew D. Blankschien, James M. Clomburg, Jacob E. Vick |
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| Rok vydání: | 2015 |
| Předmět: |
Coenzyme A
Enoyl-acyl carrier protein reductase Bacillus subtilis Reductase medicine.disease_cause Applied Microbiology and Biotechnology Catalysis chemistry.chemical_compound Escherichia coli Fatty Acid Synthase Type II medicine chemistry.chemical_classification Ecology biology Escherichia coli Proteins Fatty Acids NAD biology.organism_classification Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) Complementation Kinetics Enzyme chemistry Biochemistry Acyl Coenzyme A NAD+ kinase Oxidation-Reduction Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 81:1406-1416 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.03521-14 |
| Popis: | We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-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 [ Eg TER]) 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 β-oxidation reversal. Overexpression of FabI proved as effective as Eg TER 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 Δ fabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli . |
| Databáze: | OpenAIRE |
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