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...

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