1. 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

2. Objectives of Synthetic Biology Design modular parts to build devices and systems Metabolic engineering: enzymes are the interchangeable parts Two approaches to engineering pathways: Use naturally existing pathway Novel combination of enzymes

3. Synthetic D-Glucaric Acid Pathway Valuable chemical for industry and medicine Currently produced via non-selected and expensive oxidation process Known pathway in mammals - more than ten steps!

4. D-Glucaric Acid Recombinant System Recruited three separate enzymes from various sources in E. coli: 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

5. Optimization of Metabolic Flux Defined as the rate of turnover of molecules in a metabolic pathway Methods include modulating expression with: Promoter and ribosome binding Controlling mRNA processing rates Improving rate-limited enzymes with directed evolution Dueber, et al. Stephanopoulos & Jensen, 2005

6. Alternative - Using Synthetic Scaffolds Dueber, et al. Three domains: GBD domain recruits Udh, PDZ recruits MIOX, SH3 recruits ino1

7. Alternative - Using Synthetic Scaffolds Previous work: 4-fold increase in glucaric acid by colocalizing ino1 and MIOX in 1:1 ratio Prior observations: MIOX has lowest activity and... activity is influenced by high substrate concentration To optimize flux, improve MIOX activity

8. Alternative - Using Synthetic Scaffolds ino1 catalyzes glucose-6-P to myo -inositol, the MIOX substrate Reduce diffusion distance/time Improve effective substrate concentration, and therefore activity (and production)

9. Does MIOX activity increase alongside titer production? 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)

10. Optimizing flux by altering scaffold design Independent expression control 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: metabolic burden sequestering effect Tested GBD1 SH34 PDZ4

11. Optimizing flux by altering scaffold design Hypothesis: 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 varied ino1 between 1 and 8 varied MIOX between 1 and 4

12. Optimizing flux by altering scaffold design Titer depends on number of ino1 domains, not MIOX domains Supports consensus that substrate concentration crucial for MIOX activity

13. Optimizing flux by altering scaffold design Titer decreased as SH3 domains increased beyond 4

14. Optimizing flux by altering scaffold design Titer decreased as SH3 domains increased beyond 4

15. Optimizing flux by altering scaffold design Correlation between titer and MIOX activity across a variety of scaffolds

16. Relevance and Conclusions 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