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 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 styreneProduces medium chain length-

Polyhydroxyalkanoates (mcl-PHAs) under conditions of nitrogen limitation

styS styR styA 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 mutation2. Plate mutants on media containing Indole & Citrate3. Selection of mutants exhibiting (unrepressed) blue phenotype first4. 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 σsrpoS

DnaK supressordksA

Transcriptional Regulators

FunctionGene

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

ΔclpX and Wild Type Growth on Glucose and Citrate

WT - Citrate

Mut - Citrate

WT - Glucose

Mut - Glucose

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

11 hours post inoculation

OD

600n

m

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

ΔclpX and Wild Type Growth on PAA and Styrene

WT - PAA

Mut - PAA

WT - Styrene

Mut - Styrene

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

11 Hours post inoculation

OD

600n

m

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 IdentitiesMutant Disrupted Gene

PHA45Bacyl-CoA dehydrogenase domain protein

PHA48A

PHA30C

Calcineurin Phosphoesterase C-terminal domain proteinPHA36A

PHA43B

PHA46BdnaJ

PHA29B

PHA39BgacS

PHA45A

PHA6C/5C:1Surface adhesion protein, putative / Calcium-binding outermembrance like

protein mus24PHA46-51D

PHA6C/5C:2

PHA7F:2Transcriptional regulator - LysR family

PHA7F:2

PHA36C Transcriptional regulator, TyrR / Sigma 54 dependant transcriptional regulator PhhRPHA5B: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