Enzymatic cascade of DERA and ADH for lactone synthesis

This study presents a three-step one pot enzymatic cascade for the synthesis of a δ-lactone. Utilising acetaldehyde, combining 2-deoxyribose-5-phosphate aldolase (DERA) with an alcohol dehydrogenase (ADH) and a cofactor regeneration system this δ-lactone is synthesised with the same stereochemistry as the statin side chain precursor. The initial stage in this cascade involves the double aldol reaction, catalysed by DERA to produce the chiral lactone precursor from the achiral substrate acetaldehyde. The main challenge at this stage is the instability of DERA in the presence of high acetaldehyde concentrations. Therefore, Lactobacillus brevis DERA with a high natural acetaldehyde tolerance was genetically engineered to further improve this property. LbDERA C42M E78K exhibited improved activity and stability (no activity loss over 2 h) compared to the wild type (20% activity loss). In the second stage of the cascade, the aldol product is selectively oxidised to the lactone. A commercially available ADH was identified to selectively catalyse this oxidation using NADP⁺ as electron acceptor. NADP⁺ regeneration was achieved using O₂ as substrate in two different ways: using either photo-activated flavin or NADPH oxidase (NOX). The lactone was successfully purified from the enzymatic cascades from a preparative scale reaction in 97% purity with an optical rotation [α]_D = +34.2° (c = 0.7), proving the feasibility in a multi-enzyme three-step one-pot cascade.