Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli

Abstract

The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoC Ec gene in E. coli WB108. Also, E. coli W3110 possessing fully functional ␤-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoC Ec gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His 6 -Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the ␤-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.Polyhydroxyalkanoates (PHAs) are polyesters of (R)-hydroxyalkanoic acids accumulated in numerous bacteria as an energy and carbon storage material under nutrient limiting condition in the presence of excess carbon source (1,17,20). PHAs have been attracting much attention as they can be used as biodegradable polymers (20) and as the sources of chiral pools for the synthesis of fine chemicals (18). The metabolic pathways for the biosynthesis and degradation of PHAs have been well examined in many bacteria (17,20). For example, in short-chain-length-PHA-producing bacteria such as Ralstonia eutropha and Alcaligenes latus, two acetyl coenzyme A (acetylCoA) moieties derived from various carbon sources are condensed to acetoacetyl-CoA by 3-ketothiolase (PhaA) and sequentially converted to (R)-3-hydroxybutyryl-CoA (R3HB-CoA) by acetoacetyl-CoA reductase (PhaB). Then, R3HB-CoA is added to the growing chain of poly(3-hydroxybutyrate) [P(3HB)] by the short-chain-length PHA synthase (PhaC) (29).In pseudomonads belonging to the rRNA homology group I, the intermediates of fatty acid metabolism including enoylCoA, (S)-3-hydroxyacyl-CoA, 3-ketoacyl-CoA, and 3-hydroxyacyl-acyl carrier protein (ACP) are major precursors for medium-chain-length (MCL) PHAs (17,20,36). The metabolic links between the fatty acid metabolism and PHA biosynthesis are mediated by various enzymes such as enoyl-CoA hydratase (7,8,9,23,34,35), 3-ketoacyl-ACP reductase (22, 27, 33), epimerase (20), and 3-hydroxyacyl-ACP:CoA transacylase (12, 26). The genes encoding the...

Aerobic Benzoyl-Coenzyme A (CoA) Catabolic Pathway in Azoarcus evansii : Conversion of Ring Cleavage Product by 3,4-Dehydroadipyl-CoA Semialdehyde Dehydrogenase

“…The E. coli protein contains two domains. The C-terminal domain is an enoyl-CoA hydratase domain (28), while the N terminus shows strong similarity to BoxD and other members of the ALDH protein family. Since the aerobic pathways of benzoate degradation in A. evansii and of phenylacetate degradation in E. coli and A. evansii show strong similarities (14), it is reasonable to propose that the ALDH domain of PaaZ protein might catalyze a reaction and a substrate similar to those of BoxD.…”

“…1B). The product of orf9 shows similarity to proteins assumed to participate in the aerobic metabolism of phenylacetate in Escherichia coli (PaaZ), A. evansii (PaaZ), Pseudomonas putida (Phal), and presumably other bacteria (12,15,26,28,30). The amino acid sequence of the orf9 product shows similarity to aldehyde dehydrogenases (ALDHs).…”

The psychrophilic lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses

“…PHA compounds are of industrial interest for their thermoplastic and elastomeric properties and as sources for fine chemical synthesis (29,30). The ability of C. psychrerythraea to produce PHA compounds is likely linked with its significant capacity to produce and degrade fatty acids suggested in part by the expansion (multiple gene duplications) of acyl-CoA dehydrogenase and enoyl-CoA hydratase gene families, whose roles are central to the utilization of medium-and long-chain fatty acids that can be oxidized via the ␤-oxidation cycle and produce substrates for PHA biosynthesis (31). Because PHA composition depends in part on carbon sources and the manner in which they are catabolized (29)(30)(31), these gene family expansions may infer versatility in the nature of PHAs that C. psychrerythraea can synthesize.…”

Polyhydroxybutyrate accumulation by a Serratia sp.

“…Clearly the synthesis and production of 4-carbon units is readily achievable under conditions of excess-carbon dosing under fermentative conditions. The pathway of (anaerobic) butyrate formation could utilise several enzymes known to occur in E. coli: acetoacetyl-CoA thiolase (forms acetoacetyl-CoA from acetyl-CoA (Feigenbaum et al 1975); (this is also the first step of PHB formation)), 3-hydroxyacylCoA dehydrogenase and fructuronate reductase (convert acetoacetyl-CoA to (S)-3-hydroxy-butanoyl-CoA: (Binstock et al 1981;Pawar et al 1981;Portalier et al 1982)) and enoyl-CoA hydratase (converts (S)-3-hydroxy-butanoyl-CoA to crotonyl-CoA (Alipui et al 2002;Park et al 2003)). Further conversion to butanoyl-CoA and thence to butyrate could utilise an unknown enoyl-CoA dehydrogenase (Hoffmeister et al 2005) and, finally, acetate Co-A transferase (Korolov et al 2002).…”