BOX A2-6Controlling Metabolic Flux: Hybrid Approaches

In a hybrid rational–combinatorial approach, Dueber et al. (2009) suggested that metabolic flux could be controlled by spatially recruiting the enzymes of a desired biosynthetic pathway using synthetic protein scaffolds. To construct the enzyme scaffolding, the researchers tethered protein–protein interaction domains (for example, GBD, SH3 and PDZ domains) from metazoan signalling proteins. These domains recognize and bind cognate peptides that were fused to the enzymes to be recruited (acetoacetyl-CoA thiolase (AtoB) from Escherichia coli and HMG-CoA (HMBS) synthase and HMG-CoA reductase (HMGR) from Saccharomyces cerevisiae). By varying the number of repeats of an interaction domain, the researchers could additionally control the stoichiometry of the enzymes recruited to the complex. Using the heterologous mevalonate-dependent (MEV) pathway in E. coli as a model, they combinatorially (albeit, at a substantially smaller scale) optimized the stoichiometry of the three enzymes responsible for producing mevalonate from acetyl-CoA. Finally, they showed that the optimized synthetic scaffold could substantially increase product titre while reducing the metabolic load on the host; in other words, their high product titres did not require the overexpression of biosynthetic enzymes in the cell.