A Cell-Free Approach to Fine Chemical Pathway Engineering

Pathway engineering studies often neglect a key requirement to balance the supply and demand of enzyme loading at the molecular level. Furthermore, toxic intermediates can also hinder growth and promote genetic instability, so fine-tuning of pathway gene expression is essential to hit the metabolic sweet spot of pathway design¹.

Our aim is to draw a link between cell-free in vitro kinetics and pathway design in vivo in living cells ^2,3. Using the biosynthesis of raspberry ketone (Type III polyketide) as a 5-enzyme model pathway, we have designed a synthetic in vitro pathway to study the effects of enzyme loading and cofactor regeneration on pathway productivity. We have also discovered a reductase enzyme that fluoresces upon binding of a late pathway intermediate, therefore serving as a reporter by measuring product accumulation in real time. Using this fluorescence reporter, we have studied the pathway under massively parallel (384- to 1536-well plate format) experimental conditions with acoustic liquid handling robotics. In summary, the pathway enzyme benzalacetone synthase represents a rate-limiting step in the pathway, whilst low productivity occurs through imbalances in coenzyme A utilisation and cofactor regeneration.

Using this cell-free insight, we have implemented an in vivo pathway using combinatorial pathway refactoring⁴and natural riboswitch insulators to improve genetic stability and increase benzalacetone synthase levels. We are currently quantifying pathway designs with enzyme-GFP fusions and whole-cell extract LC-MS/MS analysis to correlate enzyme loading to pathway productivity. This will enable us to provide a fingerprint of an optimal enzyme pathway for production of Type III polyketides.

1. Jeschek, M., Gerngross, D. & Panke, S. Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort. Nat. Commun.7, 11163 (2016).

2. Weaver, L. J. et al. A kinetic-based approach to understanding heterologous mevalonate pathway function in E. coli. Biotechnol. Bioeng.112, 111â??9 (2015).

3. Opgenorth, P. H., Korman, T. P. & Bowie, J. U. A synthetic biochemistry module for production of bio-based chemicals from glucose. Nat. Chem. Biol.(2016). doi:10.1038/nchembio.2062

4. Moore, S. J. et al. EcoFlex - A Multifunctional MoClo Kit for E. coli Synthetic Biology. ACS Synth. Biol. (2016). doi:10.1021/acssynbio.6b00031