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Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3.

ABSTRACT: Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.

SUBMITTER: O'Leary ND

PROVIDER: S-EPMC1183336 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3.

O'Leary Niall D ND O'Connor Kevin E KE Ward Patrick P Goff Miriam M Dobson Alan D W AD

Applied and environmental microbiology 20050801 8

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to cl ...[more]

PMID: 16085828

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Project description:A novel strain of Pseudomonas putida LS46 was isolated from wastewater on the basis of its ability to synthesize medium chain-length polyhydroxyalkanoates (mcl-PHAs). P.putida LS46 was differentiated from other P.putida strains on the basis of cpn60 (UT). The complete genome of P.putida LS46 was sequenced and annotated. Its chromosome is 5,86,2556 bp in size with GC ratio of 61.69. It is encoding 5316 genes, including 7 rRNA genes and 76 tRNA genes. Nucleotide sequence data of the complete P. putida LS46 genome was compared with nine other P. putida strains (KT2440, F1, BIRD-1, S16, ND6, DOT-T1E, UW4, W619 and GB-1) identified either as biocontrol agents or as bioremediation agents and isolated from different geographical region and different environment. BLASTn analysis of whole genome sequences of the ten P. putida strains revealed nucleotide sequence identities of 86.54 to 97.52%. P.putida genome arrangement was LS46 highly similar to P.putida BIRD1 and P.putida ND6 but was markedly different than P.putida DOT-T1E, P.putida UW4 and P.putida W619. Fatty acid biosynthesis (fab), fatty acid degradation (fad) and PHA synthesis genes were highly conserved among biocontrol and bioremediation P.putida strains. Six genes in pha operon of P. putida LS46 showed >98% homology at gene and proteins level. It appears that polyhydroxyalkanoate (PHA) synthesis is an intrinsic property of P. putida and was not affected by its geographic origin. However, all strains, including P. putida LS46, were different from one another on the basis of house keeping genes, and presence of plasmid, prophages, insertion sequence elements and genomic islands. While P. putida LS46 was not selected for plant growth promotion or bioremediation capacity, its genome also encoded genes for root colonization, pyoverdine synthesis, oxidative stress (present in other soil isolates), degradation of aromatic compounds, heavy metal resistance and nicotinic acid degradation, manganese (Mn II) oxidation. Genes for toluene or naphthalene degradation found in the genomes of P. putida F1, DOT-T1E, and ND6 were absent in the P. putida LS46 genome. Heavy metal resistant genes encoded by the P. putida W619 genome were also not present in the P. putida LS46 genome. Despite the overall similarity among genome of P.putida strains isolated for different applications and from different geographical location a number of differences were observed in genome arrangement, occurrence of transposon, genomic islands and prophage. It appears that P.putida strains had a common ancestor and by acquiring some specific genes by horizontal gene transfer it differed from other related strains.

Project description:BackgroundEfficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy-co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440-for the first time, which simultaneously improved both cell biomass and PHA production.ResultsCo-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4-16.1% and PHA content by 29.0-63.2%, respectively, compared with feeding glycerol alone. GC-MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4-4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8-3.5%. The 1H-13C HMBC, 1H-13C HSQC, and 1H-1H COSY NMR analysis confirmed that the PHA monomers (C6-C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD+/NADH and NADPH/NADP+ ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner-Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis.ConclusionsThis work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

Dataset Information

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3.

Publications

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3.

Similar Datasets

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