increased biodegradable plastic production in pseudomonas putida ca-3 using genetic engineering...
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Increased biodegradable plastic production in Pseudomonas putida CA-3 using genetic engineering approaches
William Ryan
15/12/2010
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Identification of Potential Regulators of PHA Production
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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
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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
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GacS - Linking Pathway Activation & PHA production Currently analysing growth profiles of PHA
mutants of interest
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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
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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