YfcX Enables Medium-Chain-Length Poly(3-Hydroxyalkanoate) Formation from Fatty Acids in Recombinant Escherichia coli fadB Strains

Abstract

Expression ofEscherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHA MCL ) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional ␤-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHA MCL s when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB ؊ YfcX ؊ PhaC ؉ strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.Polyhydroxyalkanoates (PHAs) are a class of biopolyesters that are receiving considerable attention for use as renewable plastics in packaging and personal hygiene items (40). A broad spectrum of properties can be obtained from PHAs by varying the monomer unit composition (38,40). PHAs containing short-chainlength monomer units are thermoplastics, whereas PHAs containing medium-chain-length monomer units are elastomeric. While considerable progress has been made toward understanding and manipulating pathways for the formation of short-chainlength PHAs (23,34,38), such as poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate, our understanding of the biosynthesis of medium-chain-length PHAs (PHA MCL ) is not as extensive. Escherichia coli engineered with a PHA synthase possessing substrate specificity for medium-chain-length monomer units will only produce significant levels of PHA MCL when grown in the presence of fatty acids if the activity of the fatty acid ␤-oxidation complex has been disrupted by mutation or chemical inhibition (37). Since ␤-oxidation activities are required to process longer-chain fatty acids to PHA MCL monomer units (Fig. 1), the requirement of a disrupted fatty acid ␤-oxidation complex for PHA MCL formation is counterintuitive.In the study described here, we examined the ability of fadB mutants of E. coli to produce PHA MCL from longer-chain fatty acids. FadB encodes the ␣ subunit of a multienzyme complex that is involved in the degradation of fatty acids in E. coli (4). It has been previously suggested (37) that FadB activities are involved in PHA MCL formation in E. coli strain LS1298, a fadB mutant of E. coli, despite reports (9) that the strain is devoid of ␤-oxidation activities. These unexplained results encouraged us to evaluate the ability of fadB strains to produce PHA MCL from longer-chain fatty acids and to search for alternative activities that may play a role in PHA MCL formation.Searches of the E. coli nu...

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

“…On the other hand, the coexpression of the yfcX Ec and phaC2 Ps genes in E. coli WB101 resulted in the accumulation of PHA up to 0.39 g/liter, which is higher than that obtained with E. coli WB101 harboring the phaC2 Ps gene only (0.21 g/liter). The fadB yfcX mutant E. coli WB112 showed decreased PHA biosynthetic activity as previously reported by Snell et al (30). The restoration of PHA biosynthetic activity of E. coli WB112 was achieved by the plasmid-based expression of the yfcX Ec gene (Table 3).…”

“…The enzymes responsible for connecting the ␤-oxidation and the PHA biosynthetic pathways in E. coli have not been thoroughly elucidated yet. Only recently, YfcX, which is homologous to FadB, has been suggested to be responsible for supplying MCL-PHA precursors from the ␤-oxidation pathway when FadB activity is removed in recombinant E. coli (30). We, therefore examined whether the overexpression of the yfcX Ec gene can establish PHA biosynthetic pathway in E. coli W3110 harboring the Pseudomonas sp.…”

“…The enoyl-CoA hydratase activity was measured by assaying the hydration of crotonyl-CoA (Sigma Co.) at 263 nm (DU series 600 spectrophotometer; Beckman, Fullerton, Calif.) (2,8,30). A 10-l aliquot of enzyme solution was added to 90 l of 50 mM Tris-HCl (pH 8.0) containing 0.25 mM crotonyl-CoA.…”

“…There has been a report showing that the overexpression of Pseudomonas oleovorans fadBA genes could not establish the PHA biosynthetic pathway in recombinant E. coli (7). Recently, YfcX, which is homologous to FadB, was found to be necessary for the MCL-PHA formation in a fadB mutant E. coli strain (30).…”

“…Recently, another ␤-oxidation pathway operating under anaerobic conditions using nitrate as a terminal respiratory electron acceptor, which is composed of YfcYX, was found to exist besides aerobic ␤-oxidation pathway in E. coli (5). Also, it was reported that the deletion of the yfcX gene in fadB mutant E. coli abolished PHA biosynthetic capacity (30). When the fadA and/or fadB genes are deleted, other enzymes such as YfcYX, which are able to use the ␤-oxidation cycle intermediates as substrates, seem to functionally operate.…”