Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating mediumchain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogenlimited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth). INTRODUCTIONPseudomonas putida CA-3 has been reported to utilize styrene as a sole source of carbon and energy and to accumulate the biodegradable polymer medium-chainlength polyhydroxyalkanoate (mclPHA) when nitrogen (N) becomes limited in the growth medium (O'Connor et al., 1996;. These two abilities have been employed in the two-step chemo-biotechnological conversion of polystyrene, a non-biodegradable polymer, to mclPHA at laboratory scale (Ward et al., 2006). The process has been improved through the controlled feeding of N to the growth medium, which results in a twofold increase in biomass and a 1.4-fold increase in mclPHA accumulation (Goff et al., 2007).Molecular investigations of the styrene degradation pathway in this organism have been reported (Mooney et al., 2006b;O'Connor et al., 2001). Styrene metabolism in P. putida CA-3 proceeds via initial side chain oxidation and involves an upper pathway converting styrene to phenylacetic acid (PA) (O'Connor et al., 1995), and a lower pathway initiated via activation of PA to phenylace...

A fatty acyl coenzyme A synthetase (FadD) from Pseudomonas putida CA-3 is capable of activating a wide range of phenylalkanoic and alkanoic acids. It exhibits the highest rates of reaction and catalytic efficiency with long-chain aromatic and aliphatic substrates. FadD exhibits higher k cat and K m values for aromatic substrates than for the aliphatic equivalents (e.g., 15-phenylpentadecanoic acid versus pentadecanoic acid). FadD is inhibited noncompetitively by both acrylic acid and 2-bromooctanoic acid. The deletion of the fadD gene from P. putida CA-3 resulted in no detectable growth or polyhydroxyalkanoate (PHA) accumulation with 10-phenyldecanoic acid, decanoic acid, and longer-chain substrates. The results suggest that FadD is solely responsible for the activation of long-chain phenylalkanoic and alkanoic acids. While the CA-3⌬fadD mutant could grow on medium-chain substrates, a decrease in growth yield and PHA accumulation was observed. The PHA accumulated by CA-3⌬fadD contained a greater proportion of short-chain monomers than did wild-type PHA. Growth of CA-3⌬fadD was unaffected, but PHA accumulation decreased modestly with shorter-chain substrates. The complemented mutant regained 70% to 90% of the growth and PHA-accumulating ability of the wild-type strain depending on the substrate. The expression of an extra copy of fadD in P. putida CA-3 resulted in increased levels of PHA accumulation (up to 1.6-fold) and an increase in the incorporation of longermonomer units into the PHA polymer.Fatty acyl coenzyme A (CoA) synthetases (FACS; fatty acid: CoA ligases; EC 6.2.1.3) are ATP-, CoA-, and Mg 2ϩ -dependent enzymes that activate alkanoic acids to CoA esters for ␤ oxidation ( Fig. 1) (2, 17). FACS are widely distributed in both prokaryotic and eukaryotic organisms and exhibit a broad substrate specificity (34). FadD is a cytoplasmic membrane-associated FACS (7), with sizes ranging from 47 kDa to 62 kDa (2, 14). There is a lack of biochemical information on FadD with a preference for long-chain aromatic and aliphatic substrates. In the current study we purify and characterize for the first time a true long-chain FadD with activity toward both phenylalkanoic and alkanoic acids.It is known that bacteria such as Pseudomonas putida can accumulate the biological polyester polyhydroxyalkanoate (PHA) from aromatic as well as aliphatic alkanoic acids (5, 6, 42, 45). The presence of aromatic monomers in the PHA polymer suggests that a FadD with activity toward aromatic substrates is present in these PHA-accumulating strains. Garcia et al. knocked out an acyl-CoA synthetase in P. putida U with a high homology to long-chain fadD products from Escherichia coli and Pseudomonas fragi (6). Garcia et al. also showed that the mutant was not capable of growth or PHA accumulation with aromatic and aliphatic substrates having between 5 and 10 carbons in their acyl chain, indicating that it is a general and not a long-chain acyl-CoA ligase (6). In a follow-up study, Olivera et al. showed that the fadD mutants reverted to wild-t...

The improvement and modeling of a process for the supply of the volatile aromatic hydrocarbon, styrene, to a fermentor for increased biomass production of the medium chain length polyhydroxyalkanoate (mcl-PHA) accumulating bacterium Pseudomonas putida CA-3 was investigated. Fed-batch experiments were undertaken using different methods to provide the styrene. Initial experiments where styrene was supplied as a liquid to the bioreactor had detrimental effects on cell growth and inhibited PHA polymer accumulation. By changing the feed of gaseous styrene to liquid styrene through the air sparger a 5.4-fold increase in cell dry-weight was achieved (total of 10.56 g L(-1)) which corresponds to a fourfold improvement in PHA production (3.36 g L(-1)) compared to previous studies performed in our laboratory (0.82 g L(-1)). In addition this final improved feeding strategy reduced the release of styrene from the fermentor 50-fold compared to initial experiments (0.12 mL total styrene released per 48 h run). An unstructured kinetic model was developed to describe cell growth along with substrate and oxygen utilization. The formation of dispersed gas (air) and liquid (styrene) phases in the medium and the transfer of styrene between the aqueous and dispersed liquid droplet phases was also modeled. The model provided a detailed description of these phase transitions and helped explain how the feeding strategy led to improved process performance in terms of final biomass levels. It also highlighted the key factors to be considered during further process improvement.