Engineering of methionine-auxotroph via parallel evolution of two enzymes from direct-sulfurylation pathway enables its recovery in minimal medium.

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. , the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph strain (MG1655) with deletion of , encoding for L-homoserine O-succinyl transferase, and , encoding for cystathionine gamma synthase, could be complemented by introducing the genes , encoding for L-homoserine O-acetyltransferases and , encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing and from failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the and of and transformed it into a methionine-auxotrophic strain lacking the and genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant mutations in the evolved methionine-autotrophs were L315P and H46R. Interestingly, we found that a gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the methionine direct-sulfurylation pathway, leading to efficient complementation.