Engineering an Amine Transaminase for the Efficient Production of a Chiral Sacubitril Precursor

An amine transaminase was engineered for the efficient production of a chiral precursor to sacubitril, (2R,4S)-5-([1,1'-biphenyl]-4-yl)-4-amino-2-methylpentanoic acid, a key component in the blockbuster heart failure drug Entresto. Starting from an enzyme with trace activity and preference for the undesired diastereoisomer, 11 rounds of enzyme evolution were performed. The resultant variant, CDX-043, showed high productivity giving 90% conversion at 75 g/L substrate concentration with 1% enzyme loading with respect to the substrate in 24 h and without the use of an organic cosolvent. The product diastereomeric purity toward the desired (2R,4S)-stereoisomer was >99.9:0.1 d.r. This variant also exhibited high process robustness and could tolerate reaction temperatures up to 65 degrees C, isopropylamine concentrations of at least 2 M, and reaction times of at least 5 days. A structural analysis of the enzyme variants gave insight into how the mutations affected activity and selectivity. This enzyme variant allows for the efficient and cost-effective production of sacubitril at large scale.

Automated summary

The engineered amine transaminase CDX-043 showed high productivity and selectivity for the desired diastereoisomer, allowing for efficient and cost-effective large-scale production of sacubitril. The structural analysis of enzyme variants provided insight into how the mutations affected activity and selectivity. The variant exhibited high process robustness and could tolerate various reaction conditions, making it a promising candidate for industrial applications.