Transcript Increased biodegradable plastic production in Pseudomonas putida

Increased biodegradable
plastic production in
Pseudomonas putida CA-3
using genetic engineering
approaches
William Ryan
15/12/2010
Research Drivers
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Styrene extensively used in polymer production and as
solvent in polymer processing
Considerable quantities of styrene waste generated
annually
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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
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Development of suitable assay to detect catabolite repression
deficient/ reduced mutants
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Method:
1.
2.
3.
4.
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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
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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
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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
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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
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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
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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