Rational Engineering of Homoserine O-Succinyltransferase from Escherichia coli for Reduced Feedback Inhibition by Methionine

Abstract

Methionine is an essential amino acid in all living organisms and has been used in various industrial applications such as food and feed additives. However, inhibition of enzymes involved in methionine biosynthesis is considered to be a crucial bottleneck for an efficient bio-based methionine production process. Homoserine O-succinyltransferase fromEscherichia coli (EcHST) has been reported to be feedback inhibited by the final product methionine. To understand the regulation mechanism of the enzyme and generate a feedback-resistant mutant, we determined the crystal structure of EcHST and elucidated the binding site of homoserine and succinyl-CoA. The enzyme kinetic experiments of EcHST revealed that the enzyme is noncompetitively inhibited by methionine with a Ki value of 2.44 mM, and we also identified a putative inhibitor binding site located in the vicinity of the substrate binding site. We then generated the EcHSTT242A variant with reduced feedback inhibition with a Ki value of 17.40 mM.

Structural and Biochemical Studies on Product Inhibition of S-Adenosylmethionine Synthetase from Corynebacterium glutamicum

“…Finally, trace amounts of contaminants were removed by size exclusion chromatography using a Hiprep 26/60 Sephacryl S-300 HR column (GE Healthcare) equilibrated with buffer A. All purification experiments were conducted at 4 °C . The purified protein was concentrated to 38 mg/mL in buffer A. Site-directed mutagenesis experiments were performed using a Quick-Change kit (Agilent), and mutant proteins were produced using the same protocol used for the wild-type Cg MetK.…”

“…All purification experiments were conducted at 4 °C. 21 The purified protein was concentrated to 38 mg/mL in buffer A. Site-directed mutagenesis experiments were performed using a Quick-Change kit (Agilent), and mutant proteins were produced using the same protocol used for the wild-type CgMetK.…”

Engineering O‐Succinyl‐L‐Homoserine Mercaptotransferase for Efficient L‐Methionine Biosynthesis by Fermentation‐Enzymatic Coupling Route

“…L‐Methionine is the only sulfur‐containing amino acid necessary for human body, [1] which is a member of aspartate family amino acids together with L‐threonine, L‐lysine and L‐isoleucine [2] . It is widely used in food, cosmetics and medicine industries [3–4] .…”

Conversion of methionine biosynthesis inE. colifrom trans- to direct-sulfurylation enhances extracellular methionine levels

“…Efforts to produce methionine in bacteria have mainly focused on the clearance of negative regulation, controlling the metabolic flux in and out of the pathway and removing feedback inhibition of enzymes that comprise part of the biosynthesis pathway (Nakamori et al, 1999; Usuda & Kurahashi, 2005; Maier et al, 2009; Li et al, 2017; Sagong et al, 2022). For instance, disruption of metJ , which is a master regulator of the methionine pathway, together with overexpression of the genes encoding for MetA and the methionine-exporter YjeH, resulted in an approximately ten-fold improvement in the production of L-methionine in E. coli (Liu et al, 2015; Huang et al, 2017).…”