“Pathway-Pondering”Metabolic Engineering Problem

Space

June 16, 2007BioQUEST Summer Workshop

Srebrenka RobicDepartment of Biology

Agnes Scott College

Kam DahlquistDepartment of Biology

Loyola Marymount University

Classical Text Book Representation of Glycolysisfrom Alberts et al. Molecular Biology of the Cell

Balancing the check book• Carbons• ATP• NAD+/NADH

The “Two” Fates of Pyruvate from Alberts et al. Molecular Biology of the Cell

TCA Cycle

Fermentation

Struggles with Teaching Metabolism

• Memorizing steps and intermediates– Getting lost in the details

• Static pictures do not convey the dynamics of metabolic flux

• Linking metabolic pathways to each other– Anabolic and catabolic processes

• Linking metabolic pathways to other cellular processes– Regulation of gene expression

What are Your Challenges/Goals when Teaching Metabolism?

• Students do not understand resident molecule idea (sources, sinks)

• Plants have mitochondria• More than glucose metabolism• Obsessed by oxygen (Marion!)• Relative amounts and recycling (consumed vs.

recycled – catalytic amounts)• Invertebrates- diversity of metabolism• Link metabolism with evolution

(Audience responses)

Genes

Proteins

Phenotype

Molecular Biology

Biochemisty

Genetics

We Would Like to Use This ParadigmWhen Teaching Metabolism (Thanks, Brian!)

Individual,Population,Ecosystem

Learning Objectives• Energetics

– storage of energy in bonds– controlled release of chemical energy

• Oxidation/reduction– links between carbon metabolism and recycling of redox agents

• Connections and coupling of various processes– flux of chemical intermediates– connections between different pathways (anabolism and

catabolism)

• Regulation– feedback loops– subcellular location– gene regulation

• Diversity of metabolism– variation within populations – variation between species– biogeochemical cycles

Metabolic Engineering Problem Space

https://engineering.purdue.edu/ChE/Research/Biochem/Biochem-01.jpg

Who Needs a Bucket of Pyruvate?

• Food additive, nutriceutical, and a weight control supplement

• Starting material for synthesis of pharmaceutically active ingredients (amino acids, Trp, Ala, and L-DOPA)

• Starting point for other industrial fermentations

http://vitaminsbeautycare.com/images/Pyruva%20Powder.jpg

World market volume >100 tons (potential for 1000 tons) a year

Chemical versus Biological Synthesis

of PyruvateCHEMICAL SYNTHESIS:•Synthesis from tartarate (pyrolysis) involves

toxic organic solvents •Cost: $8650/ton

BIOLOGICAL SYNTHESIS•“Green synthesis”•Typically made in E. coli or Torulopsis

glabrata (yeast)•Cost: $1255/ton

Can we do better than that? Can we improve the biological production

of pyruvate?

Pathway Pondering• What do you need to know?• Is there variation from organism to organism in rates of

production of pyruvate?• Is there is an easy chemical modification of pyruvate that

sequesters it from the organism?• At what temperature/pH do you need to extract, grow culture?• Is there a way to extract without damaging organism

(recylcable and ongoing fermentation)?• If pyruvate is link in a pathway, you need to shut off the next

step, take it out of oxygen environment.• Can different pathways coming into pyruvate come in at

different rates so start with something else besides glucose?• What regulatory agency does this have to go under?

(Audience responses)

Pathway Pondering

• How is pyruvate made in E. coli?• What are some of the possible fates of

pyruvate in E. coli?• Is pyruvate production optimized in E. coli?• What steps would you modify if you wanted

to engineer an E. coli strain that makes more pyruvate?

• How would you engineer a different microorganism to produce more pyruvate?

http://karamatsu.shinshu-u.ac.jp/lab/ferment/ikeda_e2.jpg

Central Carbon Metabolism in E. coli

Causey et al. (2004) PNAS 101: 2235-2240

Thinking Like a Bioengineer

• What makes a good pyruvate producing strain?

• What parameters might you want to measure and how would you compare your strain to already existing strains?

• How might you model the cost of production?• How would you take into account the

environmental impact?• How do you engineer the strain without killing

it?

Cassey et al. Data Available for Exploration in an MS Excel File

Growth Rate versus Pyruvate Production

Growth rates of various strains

00.5

11.5

22.5

33.5

44.5

1 2 3 4 5 6 7 8 9 10

Strains

Cel

l mas

s (g

/L)

Production of pyruvate

0

100

200

300

400

500

600

700

800

Strains

Pyr

uva

te (

nM

)

Red = TC44 strain

Data from Causey et al. (2004) PNAS 101: 2235-2240 analyzed by Srebrenka

Biwer et al. (2005) Ind Eng Chem Res 44: 3124-3133

Visualizing Pathways

Pyruvate Metabolism in E. coli (KEGG)

http://www.ecocyc.org

Mutations in E. coli TC44 strain shown in GenMAPP

Genes

Proteins

Phenotype

Molecular Biology

Biochemisty

Genetics

Other Questions, Datasets, Tools

Individual,Population,Ecosystem

Other Questions, Datasets, Tools

• What are the differences between pyruvate pathways in other organisms (Saccharomyces, Lactobacilli, etc.) compared to E. coli?

• How would you engineer other organisms for pyruvate production?

• Analyzing cost and environmental impact of pyruvate synthesis

• Evolution of metabolic pathways• Metagenomics, “meta” metabolic pathways

in ecosystems, bioremediation

Metabolism & Pathway Databases

KEGG at http://www.genome.ad.jp/kegg/EcoCyc at http://ecocyc.org/

MPD at http://www.gwu.edu/~mpb/ (limited but has thermodyanmic information)

GenMAPP software at http://www.GenMAPP.org