Metabolic Engineering in Post-Genomic Era

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Microbial MetabolismMetabolic Engineering in Post-Genomic EraChing-Tsan Huang (黃慶璨)Office: Agronomy Hall, Room 111Tel: (02) 33664454E-mail: cthuang@ntu.edu.tw

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DefinitionA directed improvement of cellular properties through the modification of specific biochemical reaction with the use of recombinant DNA technology. Alteration of metabolic pathways to better understand and use cellular pathways for chemical transformation, energy transduction, and supramolecular assembly.

AimTo optimize a biotechnologically important process carried out by organisms.

DisciplineGenetics

Biochemistry Molecular BiologyPhysiology

Metabolic Engineering

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http://ocw.osaka-u.ac.jp/contents/19/Prof.%20Shimizu%201.pdf

Systems and Elements in a Cell

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ME before the Genomics Revolution

The Cell as a Production Line.

OptimizationThe flux through a metabolic pathway was thought to be controlled by rate-limiting step.

Alternation

A B C D E

E1 E2 E3 E4

mRNA1

DNA1

mRNA2

DNA2

mRNA3

DNA3

mRNA4

DNA4

D’ E’

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Metabolic flux analysis (MFA) and observability metabolic pathwayCell capability analysisOn-line metabolic flux analysisMetabolic control analysis (MCA) in complicated bionetworksExperimental determination of flux distribution by isotope labeling and trace experimentKinetic analysisIntegration of Bioinformatic data

Methodologies in Metabolic Engineering

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From Maps to Equations

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Metabolic Flux AnalysisThe calculation and analysis of the flux distribution of the entire biochemical reaction network in the system of study

http://karma.med.harvard.edu/wiki/User:JeremyZucker

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Metabolic Flux AnalysisMethodology for determining important cellular physiological characteristics from the network stoichiometryThe control of the flux tends to be distributed among multiple enzymes.

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Example of Metabolic Flux Analysis

Bioreaction Network of Central Carbon Metabolism

J. Bacteriol. 184: 152-164, 2002..

Basis of 13C Metabolic Flux Analysis

Tang et al., 2007. Nano Lett. 7(3):754-760.

Carbon Source13C1-C2-C3-C4-C5-C6

(labeled)

Intracellular fluxes

PP Pathway

Glycolysis

Measurable carbon labeling pattern of isotopomers

Amino Acids

TCA Cycle

Cell

Measurable extracellular fluxes

v1v3

v2

Metabolic Flux Distribution in Chemostat Cultures

J. Bacteriol. 184: 152-164, 2002..

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Example of ME by Alternation

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http://ocw.osaka-u.ac.jp/contents/19/Prof.%20Shimizu%201.pdf

Systems and Elements in Bioprocess

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http://ocw.osaka-u.ac.jp/contents/19/Prof.%20Shimizu%201.pdf

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http://karamatsu.shinshu-u.ac.jp/lab/ferment/index_e.htm

ME in the Post-Genomic EraThe Cell as a Production Organism.

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Genomics

Proteomics

Transcriptomics

Metabolomics

Phynotypes

The Era of OmicsWhat is possible

What appears to be happing

What makes it happen

What is happening

What is shown

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Metabolic Reconstruction from the GenomePhysiology without biochemistry

http://microgen.ouhsc.edu/s_mutans/s_mutans_pathways_large.png

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Metabolic Reconstructionht

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The Wheel of Biological Understanding

http://www.bioteach.ubc.ca/Bioinformatics/whatisbioinform/