Heterologous production of l-lysine ε-oxidase by directed evolution using a fusion reporter method

Abstract

For the heterologous production of l-lysine ε-oxidase (LodA), we constructed a new plasmid carrying LodA gene fused in-frame with an antibiotic (phleomycine) resistant gene. The new plasmid was randomly mutated and the mutated plasmids were transformed into Escherichia coli BL21 (DE3) harboring lodB, which encodes a protein (LodB) acting in posttranslational modification of LodA, and active mutants were selected by phleomycin resistance and oxidase activities. One soluble LodA variant isolated by this method contained six silent mutations and one missense mutation. At these mutation points, the codon adaptations at Lys92, Ala550, and Thr646, and the amino acid substitution at His286 to Arg contributed to the production of its functional form. The active form of LodA variant was induced by post-modification of LodB in the heterologous coexpression, and the activity increased with additional NaCl and heat treatment. This is the first report of heterologous production of LodA by random mutagenesis.

Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity

“…E . coli XL-1 Red was used for random mutagenesis of pET-22a- chmox , pET-11a- atadc , pET-11a- dmgdh , and pET-11a- dmodc as previously described 27 . E .…”

Following the Evolutionary Track of a Highly Specific l -Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes

“…L AAO (L-amino acid oxidase) is an amino acid-metabolizing enzyme that catalyzes the oxidation of the main-chain amino group of L-amino acids, thereby generating ␣-keto acid and ammonia (1). Two types of LAAOs have been reported: one is flavin adenine dinucleotide (FAD) dependent (2)(3)(4) while the other is not FAD dependent (5)(6)(7)(8). The former type of LAAO is the main target of this study.…”