MEMO: A Technique for Calculating Metabolic Modules in Production Systems Without Cells

Abstract

A viable substitute for traditional microbial fermentation methods for creating value-added products
from biological feedstocks is cell-free bioproduction systems. Finding appropriate metabolic modules
with certain characteristics is a crucial first step in creating cell-free production systems. Here, we
introduce MEMO, a brand-new computational method that supports the design of cell-free systems
in a number of ways by identifying the smallest metabolic modules with predetermined
stoichiometric and thermodynamic restrictions. Specifically, the regeneration of used cofactors (such
ATP and NAD(P)H) is a major obstacle to the long-term operation of cell-free systems. The smallest
regeneration modules that recover these cofactors with the necessary stoichiometries can be
calculated using MEMO given a production pathway with specific cofactor requirements. In order to
identify the fewest appropriate modules from a given reaction database, MEMO combines the
stoichiometric and thermodynamic restrictions into a single mixed-integer linear program. By
computing regeneration modules for the recently released synthetic CETCH cycle for in vitro carbon
dioxide fixation, we demonstrate the usefulness of MEMO. We show that MEMO can determine
multiple solutions in a reasonable amount of time in two large reaction databases (MetaCyc and
BiGG) and is very flexible in considering the various 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). Glycerol is used as the substrate in the most promising regeneration modules,
which only need eight enzymatic steps. Additionally, it is demonstrated that certain of these modules
are resistant to the oxidation of NAD(P)H and the hydrolysis of ATP, two examples of spontaneous
cofactor loss. Additionally, we show that MEMO can identify cell-free production systems that
incorporate cofactor regeneration and product synthesis. All things considered, MEMO offers a
strong technique for identifying metabolic modules and, as one specific use, for creating cell-free
production systems.