Engineering of methionine-auxotrophEscherichia colivia parallel evolution of two enzymes fromCorynebacterium glutamicum’sdirect-sulfurylation pathway enables its recovery in minimal medium

Abstract

AbstractMethionine biosynthesis relies on the sequential catalysis of multiple enzymes.Escherichia coli, 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-auxotrophE. colistrain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering theEscherichia colimethionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY fromCorynebacterium glutamicumfailed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY ofCorynebacterium glutamicumand transformed it into a methionine-auxotrophicE. colistrain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media without external methionine supplementation. The dominant metX mutations in the evolved methionine-autotrophsEscherichia coliwere L315P and H46R. Interestingly, we found that a metY 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 auxotrophEscherichia colivalidated their functionality. These results show that directed enzyme-evolution enables the fast engineering of new active variants within theEscherichia colimethionine direct-sulfurylation pathway, leading to efficient complementation.