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Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (PHAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated a phaD knockout mutant by homologous recombination. Upon disruption of the phaD gene, MCL PHA polymer accumulation was decreased. The PHA granule size was reduced, and the number of granules inside the cell was increased. Furthermore, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated proteins was changed and that the predominant protein PhaI was missing in the mutant. Complementation of the mutant with a phaDharboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully restored the granule and cell sizes. Furthermore, PhaI was attached to the granules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PHA biosynthesis. However, although its main effect seems to be the stabilization of MCL PHA granules, we found that the PhaD protein is not a major granule-associated protein and therefore might act by an unknown mechanism involving the PhaI protein.

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two-to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.Many bacteria are able to accumulate poly(3-hydroxyalkanoates) (PHAs) as carbon and energy reserves. Because of their potential use as biodegradable thermoplastics and as biopolymers that can be produced from renewable resources, PHAs have been extensively studied by academic and industrial groups (2, 6, 30). Pseudomonads synthesize mainly mediumchain-length (mcl) PHAs, which consist of monomers containing 6 to 14 carbon atoms (8,14,31). Although a few PHAs have been developed commercially and marketed (6, 11), widespread use of these polymers has been hindered by high production costs (1,19). Reduction of these costs could be achieved by several means, including increasing the product yield (19) or using transgenic plants for PHA production, provided that PHA levels can be brought to 20 to 40% of the plant dry weight (23,24,32).During the past few years we have constructed several sets of mutants of Pseudomonas putida KT2442 with the goal of altering the carbon flux towards mcl PHAs. While most of these mutants contain clearly reduced mcl PHA levels, a few have exhibited increased PHA production. Preliminary analysis of one of these mutants (P. putida KT217) showed that its glyoxylate pathway was affected.From studies on P. putida KT2442, it is known that PHA precursors can be produced via the following three main pathways: ␤-oxidation, de novo fatty acid biosynthesis, and elongation of 3-hydroxyalkanoates by acetyl coenzyme A (acetylCoA) molecules (12, 32). The ␤-oxidation pathway is...

It was shown recently that recombinant Escherichia coli, defective in the β-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303–309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinant E. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I (′thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., thebad promoter and alk promoter, for the ′thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the ′thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinantE. coli consisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinant E. coli JMU193, harboring PHA polymerase and ′thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and β-oxidation.

Rhodococcus rhodochrous PB1 was isolated from compost soil by selective culture with racemic 3-phenylbutyric acid as the sole carbon and energy source. Growth experiments with the single pure enantiomers as well as with the racemate showed that only one of the two enantiomers, (R)-3-phenylbutyric acid, supported growth of strain PB1. Nevertheless, (S)-3-phenylbutyric acid was cometabolically transformed to, presumably, (S)-3-(2,3-dihydroxyphenyl)butyric acid (the absolute configuration at the C-3 atom is not known yet) by (R)-3phenylbutyric acid-grown cells of strain PB1, as shown by 1 H nuclear magnetic resonance spectroscopy of the partially purified compound and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivative. Oxygen uptake rates suggest that either 3-phenylpropionic acid or cinnamic acid (trans-3-phenyl-2propenoic acid) is the substrate for aromatic ring hydroxylation. This view is substantiated by the fact that 3-(2,3-dihydroxyphenyl)propionic acid was a substrate for meta cleavage in cell extracts of (R)-3-phenylbutyric acid-grown cells of strain PB1. Gas chromatography-mass spectrometry analysis of trimethylsilane-treated ethyl acetate extracts of incubation mixtures showed that both the meta-cleavage product, 2-hydroxy-6-oxo-2,4-nonadiene-1,9-dicarboxylic acid, and succinate, a hydrolysis product thereof, were formed during such incubations.