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 ) Microbial biodegradation receiving interest due to cost- effectiveness 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 length- Polyhydroxyalkanoates (mcl-PHAs) under conditions of nitrogen limitation

stySstyRstyA styB styC styD styE StyR P StyE StyS Cell membrane Intracellular 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  Citrate represses sty pathway  Indole converted to indigo (blue) by styA encoded monooxygenase = reporter Method: 1. Generate Tn5 mutants – random genetic mutation 2. Plate mutants on media containing Indole & Citrate 3. Selection of mutants exhibiting (unrepressed) blue phenotype first 4. 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

Chaperone Hsp60groEL Chaperones Glyceraldehyde 3-P dehydrogenasegapA Phenylaetic acid degradation proteinpaaA β subunit of F1 ATP synthaseatpD Metabolism & Energy Production Negative regulator of sigma ErseA Regulator of sigma Drsd RNA polymerase sigma factor σ s rpoS DnaK supressordksA Transcriptional Regulators FunctionGene

P. putida CA-3 & ΔclpX Growth Profiles - Non-Pathway Substrates

P. putida CA-3 & ΔclpX Growth Profiles - Pathway Substrates

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 P. putida CA-3 & ΔclpX Growth Profiles

Identification of Potential Regulators of PHA Production

Mutants grown on liquid N-Lim media and stained with Nile Blue A fluorophore Granules visualised under fluorescence Mutant Generation & Screening Mini-Tn5 mutant library screened on Solid Nitrogen Limiting Media Mutants with reduced capacity to accumulate PHA appear less opaque #PHA45A P. putida CA-3 WT

Tn5 Disrupted Gene Sequence Identities MutantDisrupted Gene PHA45B acyl-CoA dehydrogenase domain protein PHA48A PHA30C Calcineurin Phosphoesterase C-terminal domain protein PHA36A PHA43B PHA46B dnaJ PHA29B PHA39B gacS PHA45A PHA6C/5C:1 Surface adhesion protein, putative / Calcium-binding outermembrance like protein mus24 PHA46-51D PHA6C/5C:2 PHA7F:2 Transcriptional regulator - LysR family PHA7F:2 PHA36C Transcriptional regulator, TyrR / Sigma 54 dependant transcriptional regulator PhhR PHA5B:3

GacS - Linking Pathway Activation & PHA production Currently analysing growth profiles of PHA mutants of interest

Ongoing Work Complementation of clpX and gacS mutants Assessment of changes in gene expression under repressive and non- repressive 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