Metabolic pathway design for growth-associated phenylalanine production using synthetically designed mutualism

Combination of growth-associated pathway engineering based on flux balance analysis (FBA) and adaptive laboratory evolution (ALE) is a powerful approach to enhance the production of useful compounds. However, the feasibility of such growth-associated pathway designs depends on the type of target compound. In the present study, FBA predicted a set of gene deletions (pykA, pykF, ppc, zwf, and adhE) that leads to growth-associated phenylalanine production in Escherichia coli. The knockout strain is theoretically enforced to produce phenylalanine only at high growth yields, and could not be applied to the ALE experiment because of a severe growth defect. To overcome this challenge, we propose a novel approach for ALE based on mutualistic co-culture for coupling growth and production, regardless of the growth rate. We designed a synthetic mutualism of a phenylalanine-producing leucine-auxotrophic strain (KF strain) and a leucine-producing phenylalanine-auxotrophic strain (KL strain) and performed an ALE experiment for approximately 160 generations. The evolved KF strain (KF-E strain) grew in a synthetic medium (with glucose as the main carbon source) supplemented with leucine, while severe growth defects were observed in the parental KF strain. The phenylalanine yield of the KF-E strain was 2.3 times higher than that of the KF strain.