Understanding and engineering cofactor metabolism: a quest for improved biochemical production

In the field of metabolic engineering, where cells are treated as “factories” that synthesise industrial compounds, it is essential for cell metabolism to accommodate the energy and re- dox cofactor demands of synthetic pathways. A balanced supply and consumption of ATP and NAD(P)H directly influences biotechnological performance. This study develops computational and experimental frameworks to explore how ATP and NAD(P)H limit the yield of synthetic pathways during bioproduction. Constraint-based modelling was used to develop a novel computational protocol, CBA (Co- factor Balance Assessment), which tracks how ATP and NAD(P)H contribute to cell target production, as opposed to cell maintenance, biomass and waste release, in the presence of a synthetic pathway. Using butanol pathways (a non-native product in E.coli) with varying cofactor demands, CBA discerned the network-wide effects of cofactor variations on butanol yield. Results indicate that yields could be boosted by up to 13% if the introduced pathway is balanced both in terms of energy and redox. CBA simplified cofactor balance assessments and provided insights into how to improve the efficiency of recombinant strains. Physiological and metabolic responses to cofactor perturbations were also experimentally assessed. The predominant phenotypes of strains harbouring the ATP synthase and PCK knockouts included high glycolytic flux, lower biomass and ATP. These strains were used to improve ethanol production, resulting in yields 10% and 29% higher than the WT overproducing ethanol and reaching over 70% of the theoretical maximum. The low but positive ATP yields boosted ethanol production and minimised unrestricted growth. This research posits that early-stage in silico cofactor usage profiling serves as an instrument to select better performing pathways. Significant yield improvements can be achieved experimentally with a small number of cofactor-driven modifications that reduce the waste of cofactors, illustrating the potential of these strains as platforms to improve bioproduction of cofactor-neutral or cofactor-surplus synthetic pathways.Open Acces