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

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