Fig. 1.

Strategy for exogenous substrate utilization. a Introduction of a synthetic pathway is validated in silico, tested for growth, and optimized using adaptive laboratory evolution. If growth is not possible, substrate utilization is coupled to cellular fitness under co-metabolization and optimized; otherwise, either pathway design is modified or new knockouts leading to substrate-dependent growth are tested. b Substrate (methanol) essential gene deletions are obtained by knockout of a natural catabolic pathway, thus allowing biomass formation only upon use of the synthetic methanol-dependent pathway. c Example of identified knockouts leading to methanol essentiality in the case of gluconate as a carbon source