Balancing heterologous pathway - dynamic regulation of the pathway and Proteomics/Production analysis

The mevalonate pathway (MEV) has the potential to produce biofuels such as limonene and bisabolene. The heterologous expression of MEV in E. coli removes undesired regulatory points and enables higher titers of the target terpene. However, the unregulated over-expression of enzymes leads to an accumulation of metabolites with toxic and inhibitory effects. Therefore, it is essential to coordinate the relative levels of the pathway enzymes to improve production titers. In this presentation, we show our efforts to develop tools for pathway balance, and therefore, to achieve high titer production of biofuel compounds.

We show two different approaches for balancing heterologous MEV gene expression in E.coli and improve production from 1% glucose: i) Engineer a dynamic regulation of the pathway using promoters responding to an accumulation of the toxic intermediate ii) Explore expression of the MEV pathway enzymes using targeted proteomics and its associated biofuels production to define engineering strategies; a principal component analysis (PCA) of the proteomics and the production at various conditions suggests what the more balanced pathway protein expression level is to improve the production. Indeed, extra copies of the terpene synthase and a modified top portion expression led to improved biofuels production titers over 40% compared to the unbalanced systems using the same genes.