Transcript Yuan_385_talk

Use of Modular, Synthetic Scaffolds
for Improved Production of Glucaric
Acid in Engineered E. coli
Tae Seok Moon, John E. Dueber, Eric Shiue, Kristala Prather
Metabolic Engineering, January 22 2010
Presented by Yuan Zhao
Objectives of Synthetic Biology
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Design modular parts to build devices and
systems
Metabolic engineering: enzymes are the
interchangeable parts
Two approaches to engineering pathways:
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Use naturally existing pathway
Novel combination of enzymes
Synthetic D-Glucaric Acid
Pathway
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Valuable chemical for industry and medicine
Currently produced via non-selected and
expensive oxidation process
Known pathway in mammals - more than ten
steps!
D-Glucaric Acid Recombinant
System
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Recruited three separate enzymes from
various sources in E. coli:
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myo-inositol-1-phosphate synthase (ino1) from S.
cerevisiae
myo-inositol oxygenase (MIOX) from M. musculus
uronate dehydrogenase (Udh) from P. syringae
Produced titers of ~1g/L
Moon, et al 2009
Optimization of Metabolic Flux
Dueber, et al.
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Defined as the rate of turnover of molecules in
a metabolic pathway
Methods include modulating expression with:
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Promoter and ribosome binding
Controlling mRNA processing rates
Improving rate-limited enzymes with directed evolution
Stephanopoulos & Jensen, 2005
Alternative - Using Synthetic
Scaffolds
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Dueber, et al.
Three domains:
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GBD domain recruits Udh, PDZ recruits MIOX, SH3 recruits ino1
Alternative - Using Synthetic
Scaffolds
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Previous work: 4-fold increase in glucaric acid
by colocalizing ino1 and MIOX in 1:1 ratio
Prior observations:
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MIOX has lowest activity and...
activity is influenced by high substrate concentration
To optimize flux, improve MIOX activity
Alternative - Using Synthetic
Scaffolds
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ino1 catalyzes glucose-6-P to myo-inositol, the
MIOX substrate
Reduce diffusion distance/time
Improve effective substrate concentration, and
therefore activity (and production)
Does MIOX activity increase
alongside titer production?
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Examined MIOX activity using same simple
1:1 Ino1:MIOX scaffold
Bradford assay on lysate samples to measure
activity
Scaffolded system was 19.0±0.9 nmol/min/mg,
25% higher than non-scaffolded (15.0±1.3)
Optimizing flux by altering
scaffold design
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Independent expression control
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scaffolds used tetracycline-inducible
promoter
pathway used IPTG-inducible pathway
Maximal titers at concentrations of
108nM and 0.05mM respectively
Overexpression is deleterious due
to:
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metabolic burden
sequestering effect
Tested GBD1 SH34 PDZ4
Optimizing flux by altering
scaffold design
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Hypothesis:
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Increasing ino1-recruiting domains (SH3) result in
increased MIOX activation*
Increased MIOX-recruiting domains (PDZ) may also
be important
Constructed scaffold with all three enzymes
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varied ino1 between 1 and 8
varied MIOX between 1 and 4
Optimizing flux by altering
scaffold design
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Titer depends on number of ino1 domains, not MIOX
domains
Supports consensus that substrate concentration
crucial for MIOX activity
Optimizing flux by altering
scaffold design
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Titer decreased as SH3 domains increased beyond 4
Optimizing flux by altering
scaffold design
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Titer decreased as SH3 domains increased beyond 4
Optimizing flux by altering
scaffold design
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Correlation between titer and MIOX activity across a
variety of scaffolds
Relevance and Conclusions
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Combination of foreign enzyme components to form novel
pathway
Value of metabolic flux balance in engineering
Scaffolding as an approach to improve activity
“Modularity” of enzyme recruiting domains - much variability in
affinity, etc.
Effectiveness of technique (5-fold vs 77-fold in previous
literature)
Physical constraints (orientation, sequestering) and metabolic
burden on host cells