Biologia de sistemas e o desenvolvimento de plataformas

bacterianas para bioprodutos

José Gregório Cabrera Gomez

Biology and Biotechnology

omics

Lee et al., 2005

Metabolic engineering is the improvement of cellular activities bymanipulation of enzymatic, transport, and regulatory functions ofthe cell with the use of recombinant DNA technology. Theopportunity to introduce heterologous genes and regulatoryelements distinguishes metabolic engineering from traditionalgenetic approaches to improve strains.

... An interactive cycle of a genetic change, an analysis of theconsequences, and the design of a further change...

Toward a Science of Metabolic Engineering.James E. Bailey Science, 252: 1668-1675.

Metabolic Engineering The knockout oroverexpression of genes,usually used in GeneticEngineering, frequentlydoes not result in productyield improvements due aresistance in themetabolism. Therefore, abetter knowledge of themetabolism is needed topromote metabolismengineering as a whole toimprove biotechnologicalprocesses.

Vallino & Stephanopoulos,1992

Metabolic engineering is an enabling science, and distinguishes itself fromapplied genetic engineering by the use of advanced analytical tools foridentification of appropriate targets for genetic modifications and possiblyeven the use of mathematical models to perform in silico design of optimizedcell factories. Nielsen & Jewett, 2008 FEMS Yeast Res.

analysis of cellular metabolism

Tools for analysis of cellular metabolism can be grouped into three

categories, all of them developed from the same mathematical model:

(1) Metabolic flux analysis,

(2) Flux balance analysis and

(3) Metabolic pathway analysis (Elementary mode analysis).

µ.C (negligible)

dC/dt = 0 (steady state)

S.r = 0 (Eq 2)

ri > 0 (Eq 3)

A B E F C D G H

Metabolic pathway analysis(Elementary (flux) analysis)

Elementary mode analysis calculates all solutions in the admissible

flux space by solving Eq 2 in conjunction with the thermodynamic

constraint (3) and additional non-decomposability and systematic

independence constraints. Each solution (re)presents an elementary

(flux) mode.

S.r = 0 (Eq 2) ri > 0 (Eq 3)

Metabolic models

Nielsen & Jewett, 2008

Integrating information

What are polyhydroxyalkanoates?

Bacteria rarely produces neutral lipids (triglycerides) as carbon and energy reserve.

However polyhydroxyalkanoates are produced by several bacteria.

PHA are stored in intracellular granules and can represent up to 80% of CDW.

A sink of reducing equivalents.

What are polyhydroxyalkanoates?

PHA are polyesters.

About 150 monomers were identified as constituent of PHA produced by bacteria...

...but some are inserted on PHA only if

related carbon sources are supplied.

What are polyhydroxyalkanoates?

PHA are biodegradable and biocompatible thermoplastics.

PHA as tailor-made polymers.

Naturally produced PHA – P3HB

P3HB is produced by bacteria belonging to several genera.

P3HB production in Recombinant E. coli

P3HB production in Recombinant E. coli

P3HB production in Recombinant E. coli

Dissolved Oxigen 30% saturation

Dissolved Oxigen 1% saturation

P3HB production in Recombinant E. coli

Dissolved Oxigen 30% saturation

Dissolved Oxigen 1% saturation

P3HB production in Recombinant E. coli

It had been proposed that, contrary to the natural producers, E. coli not requires nutrient limitation to produce P3HB

(Lee, 1996).However, in our laboratory, P3HB was produced by

recombinant E. coli only under linear growing conditions (O2

limited or constant feeding of a solution with defined C/N), probably due to the deficient supply of precursors for 3HB synthesis under limited nutrient conditions (Bocanegra-

Rodriguez, 2012).

P3HB production in Recombinant E. coli

P3HB = 16.9% CDW

P3HB = 50% CDW

P3HB production in Recombinant E. coli

Naturally produced PHA – P3HB-co-3HHx

P3HB-co-3HHx are produced by Aeromonas spp from plant

oils and fatty acids.

PHA production by Burkholderia sacchari

PHA production by Burkholderia sacchari

PHA production by Burkholderia sacchari

Naturally produced PHA - PHAMCL

Medium-chain-length PHA are produced mainly by

Pseudomonas spp.

Table 1. Production of PHAMCL from carbohydrates by some sugarcane soil isolates. PHA composition (mol%) Bacterial

strain

CDW

(g/L) 3HHx 3HO 3HD 3HDd

PHA

(%CDW)

YGPHA/G

(g/g)

%YMAX

KT2440 3.96 3.25 12.48 79.88 4.39 48.52 0.127 60.0

LFM046 3.72 5.01 21.85 71.83 1.31 60.51 0.161 62.9

LFM047 2.21 1.72 22.88 61.81 13.58 13.99 0.023 34.3

LFM050 2.50 0.92 15.98 67.75 15.35 18.55 0.037 41.9

LFM065 4.06 0.00 8.88 87.81 3.30 30.52 0.084 60.6 CDW – Cell dry weight 3HHx - 3-hydroxyhexanoic acid 3HO - 3-hydroxyoctanoic acid 3HD - 3-hydroxydecanoic acid 3HDd - 3-hydroxydodecanoic acid YG

PHA/G – global PHA yield from glucose %YMAX - percentual of the maximum theoretical yield.

Pseudomonas sp. LFM046Continuous culture – Steady state

VΦ

D=Φ/V Since

Φ

Thus

Sterile feending, so Xo = 0 estéril)

At steady state dX/dt = 0

OU

Growth

Growth

Fluxomics

Fluxes distribution for PHA production

by Pseudomonas sp. from glucose.

Optimal fluxes distribution for PHAproduction by Pseudomonas sp. fromglucose.

Aminoacids based analysis of fluxes using labeled carbon

Advantage

Large amounts in the cells (30-50%).

Produced from differentintermediates of the centralmetabolism (richly informative).

Disadvantage

They are not produced in largeamounts under non-growingconditions.

PHA based analysis of fluxes using labeled carbon

Advantage

Large amounts in the cells (up to80%).

It can be produced under growingor non-growing conditions.

Disadvantage

Essentially produced from acetyl-CoA (poorly informative)

Metabolic flux – Tracer experiments

Flux quantification – an optimization problem

Metabolicnetwork

2

21ˆ ,

ˆ( )min

mi i

i ix

x xθ σ=

−∑

Measured values x

Estimated values x̂

Glicose

G6P F6P

G3P

PYR

P5P

1.00

AcCoA

P5PG3P

S7P

E4P

P5P

CO2

PHA

CO2

via deEntner Doudoroff

1.443

0.481

0.962

0.519

0.481

0.481

0.481

1.519

1.519

ED pathway

Glucose

PP pathway

60

65

70

75

80

85

90

pBBR1MCS-2 eda edd eda/edd

% o

f m

axim

um

theore

tical yie

ld

Pseudomonas sp. recombinant strains

Olavarria et al,

submitted

Keasling, 2010

OverviewLaboratory of Bioproducts

OverviewLaboratory of Bioproducts

GlucoseXylose

GlycerolFat acidsPlant oilsSoybeanmolasses

Pseudomonas sp.B. sacchari

E. coli

Platforms for...

PHARhamonolipids1,3-Propanediol

Others...

Team and Financial Support

J.Gregório C. GomezLuiziana F. SilvaMarilda Keico TaciroKarel Olavarria GamezRogério S. GomesJohana K. Bocanegra-RodriguezThatiane T. MendonçaGabriela C. LozanoLinda P. Guaman BautistaLiege A. KawaiLucas Garbini CespedesDiana Carolina Tusso PizonBernardo Ferreira CamiloKelli Lopes RodriguesCesar W. Guzman MorenoRafael NahatCarlos FarjardoThandara Garcia RavelliJuliano CherixEdmar Ramos de Oliveira FilhoAline Carolina C. LemosAlexandre A. AlvesAelson L. Santos

jgcgomez@usp.br

Karen L. AlmeidaOdalys Rodriguez GamezArelis Abalos RodriguezJhoanne HansenGisele F. Bueno

MES-Cuba

NWONWONWONWO

TUDelft

Amanda B. Flora

Galo A. C. Le Roux (EP-USP)Carlos A. M. Riascos (EP-USP)

Paulo Alexandrino (André Fujita)Juliana Cardinali Rezende

Ruben Sanchez (UENF)

Andreas K. Gombert (UNICAMP)Walter M. van Gulik (TU Delft)Aljoscha Wahl (TU Delft)Reza Maleki Seifar (TU Delft)J. J. (Sef) Heijnen (TU Delft)

Muito obrigado pela Atenção!!