This project involved targeted genetic manipulation of the bacterial isolate Pseudomonas putida CA-3, in an attempt to enhance its ability to degrade the toxic pollutant styrene to carbon dioxide and water. The styrene degradation pathway has been well-characterised in this strain at both the genetic and physiological level. In addition, this strain has the novel ability to generate polyhydroxyalkanoate (PHA), a bio-degradable polymer, from styrene under appropriate growth conditions. Polyhydroxyalkanoates have been recognised as a potential bio-plastic replacement for current petrochemical plastics. Thus, P. putida CA-3 not only offers a significant styrene remediation capability but also the opportunity to use a toxic waste compound as the starting material for the production of value-added environmentally friendly bio-plastic. This project has focused mainly on the styrene degrading ability of P. putida CA-3, where styrene is degraded via phenylacetic acid, and seeks to develop the biotechnological potential of this strain by applying recombinant DNA strategies in an attempt to enhance the rate of styrene degradation. A considerable amount of data exists on the ability of bacteria to degrade styrene and a number of aspects of the styrene degradation pathway have been targeted in this study. Advanced molecular biology techniques including binding site mutagenesis, tri-parental mating, gene over-expression and random mutagenesis were used to generate recombinant strains that were subsequently monitored for changes in the styrene degradation pathway. When the presence of styrene is detected by the bacterial cell the styrene degradation genes are activated and converted to proteins. These proteins facilitate the initial step-wise degradation of styrene to phenylacetic acid. However, inhibitor compounds, particularly other carbon sources, can block transcription of the styrene pathway genes, thus disrupting degradation, and this could be a significant problem when trying to treat wastes since these are unlikely to consist of pure styrene. Manipulating the genetic information to block the interference from inhibitory compounds is a potential way of maintaining styrene degradation in mixed wastes. The recombinant strains were tested under a range of metabolic conditions and the genetic manipulations performed in this study did not appear to have a significant impact in P. putida CA-3, however further analysis involving mixed substrates is required. The preliminary data suggests that these inhibitor sites may play a limited role in styrene degradation in P. putida CA-3. For bacteria to successfully degrade many compounds they must be transported across the cell membrane into the cell, where they are then degraded by cellular enzymes. Transport of styrene across the bacterial cell membrane presents a limiting factor in the rate of styrene degradation and increasing the number of copies of the transport gene was seen as a potential way of increasing the rate of styrene degradation. Similarly possessing additional copies of the styrene catabolic genes may lead to greater levels of enzyme activity and thus to increased styrene degradation. A number of recombinant strains were generated and analysis suggests that non-styrene controlled expression of the genes and multiple copies are required to significantly increase styrene degradation. In P. putida CA-3 phenylacetic acid is a key intermediate in styrene degradation. Previous investigation of phenylacetic acid breakdown in this strain identified a critical gene involved in phenylacetic acid metabolism which did not share significant similarity with reported phenylacetic acid associated genes. This suggested that novel single copy genes are important for phenylacetic acid degradation in this strain. Further investigation of P. putida CA-3 resulted in the identification of a novel mutant which had lost the ability to grow with phenylacetic acid as a sole carbon source. Additional analysis of the mutant strain revealed a link between the disrupted gene and transport of phenylacetic acid into the cell. This further highlights the critical role that transport systems play in the effective operation of degradation pathways for aromatic compounds.

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O'Mahony, M. O?Leary, D.N.   "Recombinant DNA approaches to enhance the activity of the pathway for the degradation of the toxic pollutant styrene in the bioreactor isolate Pseudomonas putida CA-3: a biotechnologically significant metabolic route.". Associated datasets and digitial information objects connected to this resource are available at: Secure Archive For Environmental Research Data (SAFER) managed by Environmental Protection Agency Ireland https://eparesearch.epa.ie/safer/resource?id=dfe42489-d8e3-102e-a0a4-f81fb11d7d1c (Last Accessed: 2024-09-19)

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SAFER-Data Display URL	https://eparesearch.epa.ie/safer/iso19115/display?isoID=209
Resource Keywords	Styrene, phenylacetic acid, waste remediation, biodegradation, biotransformation, polyhydroxyalkanoates, bio-plastics, Pseudomonas.
EPA/ERTDI/STRIVE Project Code	2007-FS-ET-9-M5
EPA/ERTDI/STRIVE Project Theme	Environmental Technologies
Resource Availability:	Public-Open
Limitations on the use of this Resource	Any attached datasets, data files, or information objects can be downloaded for further use in scientific applications under the condition that the source is properly quoted and cited in published papers, journals, websites, presentations, books, etc. Before downloading, users must agree to the "Conditions of Download and Access" from SAFER-Data. These appear before download. Users of the data should also communicate with the original authors/owners of this resource if they are uncertain about any aspect of the data or information provided before further usage.
Number of Attached Files (Publicly and Openly Available for Download):	2
Project Start Date	Monday 1st October 2007 (01-10-2007)
Earliest Recorded Date within any attached datasets or digital objects	Monday 1st October 2007 (01-10-2007)
Most Recent Recorded Date within any attached datasets or digital objects	Tuesday 12th April 2011 (12-04-2011)
Published on SAFER	Thursday 26th May 2011 (26-05-2011)
Date of Last Edit	Thursday 26th May 2011 at 13:42:12 (26-05-2011)
Datasets or Files Updated On	Thursday 26th May 2011 at 13:40:15 (26-05-2011)

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