In silico design of anaerobic growth-coupled product formation in Escherichia coli: experimental validation using a simple polyol, glycerol.

Integrated approaches using in silico model-based design and advanced genetic tools have enabled efficient production of fuels, chemicals and functional ingredients using microbial cell factories. In this study, using a recently developed genome-scale metabolic model for Escherichia coli iJO1366, a mutant strain has been designed in silico for the anaerobic growth-coupled production of a simple polyol, glycerol. Computational complexity was significantly reduced by systematically reducing the target reactions used for knockout simulations. One promising penta knockout E. coli mutant (E. coli ΔadhE ΔldhA ΔfrdC ΔtpiA ΔmgsA) was selected from simulation study and was constructed experimentally by sequentially deleting five genes. The penta mutant E. coli bearing the Saccharomyces cerevisiae glycerol production pathway was able to grow anaerobically and produce glycerol as the major metabolite with up to 90% of theoretical yield along with stoichiometric quantities of acetate and formate. Using the penta mutant E. coli strain we have demonstrated that the ATP formation from the acetate pathway was essential for growth under anaerobic conditions. The general workflow developed can be easily applied to anaerobic production of other platform chemicals using E. coli as the cell factory.