The role of the fatty acid Beta-oxidation multienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoate biosynthesis: Molecular characterization of the fadBA operon from P. oleovorans and of the enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida

Abstract

In order to investigate the role of the putative epimerase function of the β-oxidation multienzyme complex (FadBA) in the provision of (R)-3-hydroxyacyl-CoA thioesters for medium-chain-length polyhydroxyalkanoate (PHAMCL) biosynthesis, the fadBA Po operon of Pseudomonas oleovorans was cloned and characterized. The fadBA Po operon and a class-II PHA synthase gene of Pseudomonas aeruginosa were heterologously co-expressed in Escherichia coli to determine whether the putative epimerase function of FadBAPo has the ability to provide precursors for PHA accumulation in a non-PHA-accumulating bacterium. Cultivation studies with fatty acids as carbon source revealed that FadBAPo did not mediate PHAMCL biosynthesis in the E. coli wild-type strain harboring a PHA synthase gene. However, PHA accumulation was strongly impaired in a recombinant E. coli fadB mutant, which harbored a PHA synthase gene. These data indicate that in pseudomonads FadBA does not possess the inherent property, based on a putative epimerase function, to provide the (R)-enantiomer of 3-hydroxyacyl-CoA efficiently and that other linking enzymes are required to efficiently channel intermediates of β-oxidation towards PHAMCL biosynthesis. However, the phaJ gene from P. oleovorans and from Pseudomonas putida, both of which encoded a 3-Re enoyl-CoA hydratase, was identified. The co-expression of phaJ Po/Pp with either a class-II PHA synthase gene or the PHA synthase gene from Aeromonas punctata in E. coli revealed that PhaJPo/Pp mediated biosynthesis of either PHAMCL, contributing to about 1% of cellular dry mass, or of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), contributing to 3.6% of cellular dry mass, when grown on decanoate. These data indicate that FadBAPo does not mediate the provision of (R)-3-hydroxyacyl-CoA, which resembles FadBA of non-PHA-accumulating bacteria, and that 3-Re enoyl-CoA hydratases are required to divert intermediates of fatty acid β-oxidation towards PHA biosynthesis in P. oleovorans.