Statins are a group of drugs used for lowering cholesterol levels in the human bloodstream to prevent cardiovascular diseases and its synthesis is a challenge because of chemical and stereochemical requirements. In this work, a multi-enzyme one-pot sequential strategy was employed to obtain lactonized statin side-chain precursor and was compared to the two-pot process. The enzymatic production of this valuable product is rarely examined comparing to the traditional chemical methods. The examined cascade consisted of two consecutive reactions: lactol production by the 2-deoxyribose-5-phosphate aldolase (DERA)-catalysed double aldol addition of acetaldehyde and chloroacetaldehyde and its oxidation by the NAD-dependent aldehyde dehydrogenase (AlDH). NADH oxidase (NOX) was employed for the coenzyme regeneration. The insight in all reactions of the system was performed by enzyme kinetic and stability studies. Based on these data the mathematical models were developed and validated in a batch reactor. The oxidation was carried out using both purified (two-pot) and crude lactol (one-pot) obtained in the DERA-catalysed reaction. Better process metrics and greater stability of the AlDH was observed in one-pot system, thus demonstrating an additional benefit in favor of running multi-enzymatic (cascade) processes. (c) 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.