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Biología sintética y de sistemas de microorganismos para la
síntesis de biomoléculas
Dr. Ignacio Poblete
Biosystems Engineering Laboratory
Center for Bioinformatics and Integrative Biology
• 46.000 Estudiantes
• La Facultad de Ciencias Biológicas cuenta con 2 programas de Doctorado
• La Universidad publica más de 500 articulos ISI por año
www.unab.cl
Synthesis of bio-based chemicals
Systems biology
• Biopolymers • Biofuels • Proteins • Nanoparticles
• Pathogen-plant Interaction
• Omics analysis
• Genome-scale modeling of microorganisms
www.cbib.cl
AIM
S R
ES
ULT
S
OU
TL
INE
S
PO
LYH
YD
RO
XY
AA
LK
AN
OA
Te
TH
E P
RO
BL
EM
Plastics
• Contribution to global warming (CO2)
• Non-renewable
• Persistent in the environment
Biopolymers
AIM
S
RE
SU
LTS
OU
TL
INE
S
PO
LYH
YD
RO
XY
AA
LK
AN
OA
TE
TH
E P
RO
BL
EM
Pseudomonas putida Recombinant E. coli Ralstonia Eutropha
• Carbon excess
• Nutrient limitation (N, P, O2)
scl (PHB, PHV)
mcl-PHA
Advanced engineering material, 2005. 7
Poly(3-hydroxypropionate) Poly(3-hydroxyalkanoate) with R=alkyl or functional groups
Poly(3-hydroxybutyrate)
or
P(3HB)
Poly(3-hydroxyvalerate)
or
P(3HV)
Biopolymers
Cupriavidus Necator Pseudomonas putida
Polyhydroxyalkanoates
Sustainable
Renewable Carbon Substrates
• Gram-negative bacterium
• High metabolic versatility
• Certified as Biosafety
• Its genome has been sequenced (2002)
Pseudomonas putida KT2440
Poblete-Castro et al. Applied Microbiology
and Biotechnology 2012, 93:2279-2290.
Poblete-Castro et al. Applied Microbiology and Biotechnology 2012, 93
SY
ST
EM
S B
IOT
EC
HN
OL
OG
Y
Networks Models Quantitative measurements
Dynamic processes
Nelson et al, 2002
Genome-scale metabolic models
Oberhardt et al, 2009
Transcriptomics Proteomics Metabolomics Fluxomics
SY
ST
EM
S B
IOT
EC
HN
OL
OG
Y
Bruggeman et al, 2006
2-keto- glutarate
acetyl-CoA
citrate
isocitrate succinate
CO2
CO2
fumarate
malate
oxaloacetate
glyoxylate
pyruvate
Acyl-ACP
Enoyl-ACP
(S)-3-hydroxyacyl-ACP
3-ketoacyl-ACP
Malonyl-CoA
Malonyl-ACP
glyceraldehyde-3P
dihydroxyacetone-P
GLUCOSE
(R)-3-hydroxyacyl-CoA
gluconate
gluconate Periplasm GLUCOSE 2-ketogluconate
2-ketogluconate
PHA
Fatty acids biosynthesis
TCA
glucose
glucose-6-P gluconate-6-P 2-ketogluconate-6-P
fructose-6-P
fructose-1,6-DP
2-keto-3-deoxy-6-P-gluconate
glycerate-3P
phosphenol- pyruvate
D-ribulose-5-P
D-ribose-5-P
Phosphoribosyl pyro-P
His-L
Xylulose-5-P
Erythrose-4-P
seduheptulose-7-P
P. putida KT2440 In-silico modeling
FBA Overview
p1
p2
p3
p4
flux v1
flux v2
flux v3
p1
p2
p3
p4
flux v1
flux v2
flux v3
p1
p2
p3
p4
flux v1
flux v2
flux v3
p1
p2
p3
p4
flux v1
flux v2
flux v3
Stoichiometric Matrix Gene annotation
Enzyme and reaction catalog
Feasible Space S*v=0
Add constraints:
vi>0 ai>vi>bi
Optimal Flux Growth objective
Z=c*v
Solve with linear programming
Flux solution
2-keto- glutarate
acetyl-CoA
citrate
isocitrate succinate
CO2
CO2
fumarate
malate
oxaloacetate
glyoxylate
pyruvate
Acyl-ACP
Enoyl-ACP
(S)-3-hydroxyacyl-ACP
3-ketoacyl-ACP
Malonyl-CoA
Malonyl-ACP
glyceraldehyde-3P
dihydroxyacetone-P
GLUCOSE
(R)-3-hydroxyacyl-CoA
gluconate
gluconate Periplasm GLUCOSE 2-ketogluconate
2-ketogluconate
PHA
Fatty acid biosynthesis
TCA
glucose
glucose-6-P gluconate-6-P 2-ketogluconate-6-P
fructose-6-P
fructose-1,6-DP
2-keto-3-deoxy-6-P-gluconate
glycerate-3P
phosphenol- pyruvate
D-ribulose-5-P
D-ribose-5-P
Phosphoribosyl pyro-P
His-L
Xylulose-5-P
Erythrose-4-P
seduheptulose-7-P
∆PP1023-1444 PP1444
PP1023 Flux ({vi}) Number of molecules per unit time through each reaction (mmol gcw-1h-1)
0 10 20 30 400
20
40
60
80
100
P
roduction flu
x o
f glu
conate
[%
]
Production flux of C10 PHA monomer [%]
0 10 20 30 400
50
100
150
200
250
(B)
Glu
cose 6
-phosphate
dehydro
genase flu
x [%
]Production flux of C10 PHA monomer [%]
(A)
Poblete-Castro et al. Metabolic Engineering 2013, 15
∆gcd accumulates 60% more PHA than the WT
∆pgl-gcd accumulates 53% more PHA than the WT
2-keto- glutarate
acetyl-CoA
citrate
isocitrate succinate
CO2
CO2
fumarate
malate
oxaloacetate
glyoxylate
pyruvate
Acyl-ACP
Enoyl-ACP
(S)-3-hydroxyacyl-ACP
3-ketoacyl-ACP
Malonyl-CoA
Malonyl-ACP
glyceraldehyde-3P
dihydroxyacetone-P
GLUCOSE
(R)-3-hydroxyacyl-CoA
gluconate
gluconate Periplasm GLUCOSE 2-ketogluconate
2-ketogluconate
PHA
Fatty acid biosynthesis
TCA
glucose
glucose-6-P gluconate-6-P 2-ketogluconate-6-P
fructose-6-P
fructose-1,6-DP
2-keto-3-deoxy-6-P-gluconate
glycerate-3P
phosphenol- pyruvate
D-ribulose-5-P
D-ribose-5-P
Phosphoribosyl pyro-P
His-L
Xylulose-5-P
Erythrose-4-P
seduheptulose-7-P
∆gcd:acoA gcd
acoA
Overproduction of AcoA and Pgl in P. putida wild-type and Δgcd
A) Overproduction of AcoA and Pgl in KT2440 wild-type
B) Overproduction of AcoA and Pgl in Δgcd
Borrero-de Acuña JM et al. 2014
PHA production enhancement in metabolically engineered strains
A) PHA content Minimal medium B) Cell dry weight Ammonium limitation & glucose as carbon source
Borrero-de Acuña JM et al. 2014
PHA production enhancement in metabolically engineered strains
A) PHA content Minimal medium B) Cell dry weight Ammonium limitation & glucose as carbon source
Borrero- de Acuña JM et al. 2014
KT2440 Δgcd overexpressing acoA increased PHA yield by 120%
Synthetic Biology
A Novel Autolysis Cell System via Synthetic Biology
Highly valuable industrial products
Polyhydroxyalkanoates
Proteins
Nanoparticles
Extraction
processes pose several drawbacks:
Enzyme cost
High temperatures
Use of expensive solvents/detergents
Lysozyme course of action
Production of mcl-PHA in P. putida KT2440 using frying oil as carbon source
Dead/alive assessment of the recombinant strain vs wild type
Dead/alive assessment of the recombinant strain vs wild type
Biosystems Engineering Lab
Thank you very much
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