Abstract
Cell-free bioproduction systems represent a promising alternative to classical microbial fermentation processes to synthesize value-added products from biological feedstocks. An essential step for establishing cell-free production systems is the identification of suitable metabolic modules with defined properties. Here we present MEMO, a novel computational approach to find smallest metabolic modules with specified stoichiometric and thermodynamic constraints supporting the design of cell-free systems in various regards. In particular, one key challenge for a sustained operation of cell-free systems is the regeneration of utilized cofactors (such as ATP and NAD(P)H). Given a production pathway with certain cofactor requirements, MEMO can be used to compute smallest regeneration modules that recover these cofactors with required stoichiometries. MEMO incorporates the stoichiometric and thermodynamic constraints in a single mixed-integer linear program, which can then be solved to find smallest suitable modules from a given reaction database. We illustrate the applicability of MEMO by calculating regeneration modules for the recently published synthetic CETCH cycle for in vitro carbon dioxide fixation. We demonstrate that MEMO is very flexible in taking into account the diverse constraints of the CETCH cycle (e.g., regeneration of 1 ATP, 4 NADPH and of 1 acetyl-group without net production of CO(2) and with permitted side production of malate) and is able to determine multiple solutions in reasonable time in two large reaction databases (MetaCyc and BiGG). The most promising regeneration modules found utilize glycerol as substrate and require only 8 enzymatic steps. It is also shown that some of these modules are robust against spontaneous loss of cofactors (e.g., oxidation of NAD(P)H or hydrolysis of ATP). Furthermore, we demonstrate that MEMO can also find cell-free production systems with integrated product synthesis and cofactor regeneration. Overall, MEMO provides a powerful method for finding metabolic modules and for designing cell-free production systems as one particular application.