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Increased biodegradable plastic production in Pseudomonas putida CA-3 using genetic engineering approaches William Ryan 15/12/2010 Research Drivers Styrene extensively used in polymer production and as solvent in polymer processing Considerable quantities of styrene waste generated annually 33 million pounds in the US alone (US TRI - 2008) Microbial biodegradation receiving interest due to costeffectiveness and environmental sensitivity Since 1998 legislation has been introduced to encourage waste reduction and environmentally conscious management Pseudomonas putida CA-3 & Styrene Pseudomonas putida CA-3 capable of degrading styrene via sty pathway P. putida CA-3 also possesses the ability to produce a biodegradeable bioplastic from styrene Produces medium chain lengthPolyhydroxyalkanoates (mcl-PHAs) under conditions of nitrogen limitation Cell membrane Intracellular styS styR styA styB styC styD styE StyS StyR StyE P Overview of sty pathway activation and degradation of styrene Degradation • StyS, StyR activation and StyE overexpression previously investigated • Current investigation focuses on potential global regulatory influences Identification of Potential sty Pathway Regulators Development of suitable assay to detect catabolite repression deficient/ reduced mutants Method: 1. 2. 3. 4. Citrate represses sty pathway Indole converted to indigo (blue) by styA encoded monooxygenase = reporter Generate Tn5 mutants – random genetic mutation Plate mutants on media containing Indole & Citrate Selection of mutants exhibiting (unrepressed) blue phenotype first Sequence area of Tn5 insertion for identification of potential regulatory elements Screening of Mutant Library highlighted mutant of interest ΔclpX ClpX ClpX is a chaperone which works in conjunction with ClpP protease to degrade many proteins ClpX works by unfolding the protein and feeding it into the ClpP for degradation Gene Function Transcriptional Regulators dksA DnaK supressor rpoS RNA polymerase sigma factor σs rsd Regulator of sigma D rseA Negative regulator of sigma E Metabolism & Energy Production atpD β subunit of F1 ATP synthase paaA Phenylaetic acid degradation protein gapA Glyceraldehyde 3-P dehydrogenase Chaperones groEL Chaperone Hsp60 P. putida CA-3 & ΔclpX Growth Profiles - Non-Pathway Substrates ΔclpX and Wild Type Growth on Glucose and Citrate 1.400 Mut - Glucose 1.200 WT - Glucose 1.000 OD600nm 0.800 WT - Citrate 0.600 0.400 Mut - Citrate 0.200 0.000 11 hours post inoculation P. putida CA-3 & ΔclpX Growth Profiles - Pathway Substrates ΔclpX and Wild Type Growth on PAA and Styrene 0.900 0.800 WT - PAA 0.700 WT - Styrene OD600nm 0.600 Mut - Styrene 0.500 Mut - PAA 0.400 0.300 0.200 0.100 0.000 11 Hours post inoculation P. putida CA-3 & ΔclpX Growth Profiles Carbon utilization is affected in clpX deficient mutants in a substrate dependant fashion Substrate transport mechanisms may be involved in the control of carbon utilisation by ClpX Identification of Potential Regulators of PHA Production Mutant Generation & Screening Mini-Tn5 mutant library screened on Solid Nitrogen Limiting Media Mutants with reduced capacity to accumulate PHA appear#PHA45A less opaque P. putida CA-3 WT Mutants grown on liquid N-Lim media and stained with Nile Blue A fluorophore Granules visualised under fluorescence Tn5 Disrupted Gene Sequence Identities Mutant PHA45B PHA48A Disrupted Gene acyl-CoA dehydrogenase domain protein PHA30C PHA36A Calcineurin Phosphoesterase C-terminal domain protein PHA43B PHA46B PHA29B PHA39B PHA45A PHA6C/5C:1 PHA46-51D PHA6C/5C:2 PHA7F:2 PHA7F:2 PHA36C PHA5B:3 dnaJ gacS Surface adhesion protein, putative / Calcium-binding outermembrance like protein mus24 Transcriptional regulator - LysR family Transcriptional regulator, TyrR / Sigma 54 dependant transcriptional regulator PhhR GacS - Linking Pathway Activation & PHA production ΔgacS & Wild Type Growth Profile 0.900 0.800 Currently analysing growth profiles of PHA mutants of interest WT - PAA WT - Citrate 0.700 WT - Styrene 0.600 OD600nm Mut - Citrate 0.500 0.400 Mut - PAA 0.300 0.200 0.100 0.000 11 hours post Inoculation Mut - Styrene Ongoing Work Complementation of clpX and gacS mutants Assessment of changes in gene expression under repressive and nonrepressive conditions Investigation of pha gene expression in PHA mutants Acknowledgements Prof. Alan Dobson Dr. Niall O’Leary Dr. Mark O’Mahony Claire Clancy Everyone in the Lab & E.R.I. Thanks to EPA for funding the research