Title: Coupling carboxylic acid reductase to inorganic pyrophosphatase enhances cell-free in vitro aldehyde biosynthesis
Abstract: Carboxylic acid reductases (CARs) have been harnessed in metabolic pathways to produce aldehydes in engineered organisms. However, desired aldehyde products inhibit cell growth and limit product titers currently achievable from fermentative processes. Aldehyde toxicity can be entirely circumvented by performing aldehyde biosynthesis in non-cellular systems. Use of purified CARs for preparative-scale aldehyde synthesis has been limited by in vitro turnover of model CARs, such as CarNi from Nocardia iowensis, despite robust conversion of substrates associated with expression in heterologous hosts such as E. coli and yeast. In this study, we report that in vitro activity of CarNi is inhibited by formation of the co-product pyrophosphate, and that pairing of an inorganic pyrophosphatase (PpaEc) with CarNi substantially improves the rate and yield of aldehyde biosynthesis. We demonstrate that, in the presence of PpaEc, Michaelis–Menten kinetic models based on initial rate measurements accurately predict CarNi kinetics within an in vitro pathway over longer timescales. We rationalize our novel observations for CarNi by examining previously posed arguments for pyrophosphate hydrolysis made in the context of other adenylate-forming enzymes. Overall, our findings may aid in increasing adoption of CARs for cell-free in vitro aldehyde biosynthetic processes.