Integrated Genomics for Enzyme and Pathway Optimization and Stability

The engineering of microbial strains with synthetic genes and pathways is often hindered by the fact that cellular metabolism and regulation is complex and small perturbations of this context can impact the function of the cell or the synthetic genes. For example, modifications of microbial central metabolism for increasing cellular levels of NAD(P)H cofactors, such as a deletion of the gene encoding phosphoglucoisomerase (pgi), can cause deleterious growth defects and impact their application in metabolic engineering.

Our study presents an integrated genomics approach to improve rational and intuitive cell factory engineering via whole-genome screening and metabolic modeling, and pathway regulation and integration via synthetic molecular regulators. We apply this approach to identify cellular functions that directly influence the availability of NADPH and the yield and stability of a 7-gene biosynthetic pathway for the production of the vitamin Thiamine. NADPH is a crucial cofactor in reductive enzymatic reactions to synthesize valuable bioactive compounds

We show that our methodology, based on the screening of a very large number of barcoded transposon insertion mutants (~10⁶) coupled with product biosensors and synthetic genes expressed from plasmids, allows a direct identification of key genes whose deletion improves performance during NADPH consumption and increases pathway stability and yield during vitamin production. We identified a number of deletions of oxidoreductases which we confirm lead to an increased NADPH/NADP⁺ ratio ranging from 12 to 62% compared to a wild type strain. We further constructed and applied a RNA riboswitch-based molecular biosensor for specific vitamins to select mutant genotypes with better growth and higher stability of vitamin production.

We are continuing to validate the role of identified cellular factors, enzymes and metabolic fluxes to build a system-wide map of cellular redox capabilities and pathway stability to assist targeted and robust metabolic engineering.