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 CO2 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.
Highlights
Cell-free bioproduction systems represent a promising alternative to classical microbial fermentation processes to synthesize value-added products from biological feedstocks
MEMO has many possible applications, we focused on its use for finding regeneration modules for cell-free production systems
As exemplified with the CETCH cycle, MEMO is very flexible in taking into account diverse requirements and is able to enumerate multiple solutions in reasonable time in very large universal networks
Summary
Cell-free bioproduction systems represent a promising alternative to classical microbial fermentation processes to synthesize value-added products from biological feedstocks. Regarding regeneration of NAD(P)H, Schwander et al.[11] used formate as an electron donor to replenish the NADPH pool via a formate dehydrogenase While this one-step module is simple and cheap and was fully sufficient to demonstrate the CO2-fixing capability of the synthetic CETCH cycle, it would, in a realistic application, negatively affect the overall stoichiometry of CO2 fixation because CO2 is released as side product of the formate dehydrogenase reaction (if formate is produced electrochemically (from CO2), its use would be carbon neutral). These examples underline the necessity of a systematic approach to identify suitable regeneration modules for cell-free production systems. Constraint (2) renders the problem significantly harder but ensures that malate is the only organic product of the CETCH cycle when being coupled with the regeneration module, which will simplify downstream processing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
