Sequence‐Guided Redesign of an Omega‐Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines

• Published in: Chembiochem : a European journal of chemical biology 2024-07, Vol.25 (14), p.e202400285-n/a
• Main Authors: Xu, Zhexian, Xu, Jiaqi, Zhang, Tao, Wang, Ziyuan, Wu, Jianping, Yang, Lirong
• Format: Article
• Language: English
• Subjects: Amines; Asymmetric synthesis; Asymmetry; Bacillus megaterium; Biocatalysts; Catalytic activity; Chemical synthesis; chiral amine; distant site; Industrial applications; Industrial production; Ketones; Mutation; Nucleotide sequence; Protein engineering; Redesign; substrate tunnel; Substrates; Thermal stability; Transaminase; Transaminases; transamination; Tunnels; ω-transaminase
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202400285

ω‐Transaminases (ω‐TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non‐native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate‐binding pockets of ω‐TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω‐TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7‐fold higher specific activity and a 19.9‐fold longer half‐life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S‐methoxyisopropylamine (S‐MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω‐TAs. The ω‐TA from Bacillus megaterium (BmeTA) is selected for engineering to catalyze the synthesis of (S)‐methoxyisopropylamine from methoxyacetone. An engineered ω‐TA (2M) with improvement of solubility, activity and thermostability is obtained through multiple sequence alignment. Furthermore, Molecule Dynamics simulation explains the mechanisms of its improved properties. 2M is also found to be more advantageous in catalyzing various ketones.