Biosynthesis of acyl-CoA thioesters from simple amines, alcohols and esters enabled by co-immobilized multi-enzyme systems

Acyl-Coenzyme A derivatives are a valuable family of metabolic intermediates with many applications, such as building blocks on biosynthetic routes, protein labeling, or potential therapeutics. However, their synthesis is limited to highly activated molecules (i.e. anhydrides) or carboxylic acids. In this work, we developed two multifunctional heterogeneous biocatalysts, capable of synthesizing acyl-CoA thioesters from primary alcohols or amines using free Coenzyme A via two different stepwise enzymatic cascades. The first enzymatic cascade involves an alcohol and CoA-acylating decarboxylase that catalyzes the oxidative thioesterification of primary alcohols through consecutive oxidation of alcohols to thioesters. In the second cascade, a transaminase enables the deamination of primary amines to aldehydes, which are concurrently oxidized to acyl-CoAs by a CoA-acylating dehydrogenase, using pyruvate as the amine acceptor. In both cases, the redox cofactor (NAD+) regeneration is carried out with a water-forming NADH oxidase that thermodynamically drives the reaction. Both systems exhibited broad substrate scopes, accepting a variety of aliphatic and aromatic primary amines and alcohols. Through rational co-immobilization designs coupled to post-immobilization steps using glutaraldehyde, we achieved highly active and operationally stable multifunctional heterogeneous biocatalysts. Specifically, the system to transform propyl amine into propyl-CoA exhibited excellent performance over five consecutive reaction cycles. Finally, the oxidative thioesterification cascade was successfully coupled with the immobilized promiscuous lipase Novozyme 435, allowing the hydrolysis of the one-pot transformation of alkyl esters into acyl-CoA thioesters. These findings broaden the range of non-activated substrates for acyl-CoA biosynthesis beyond conventional carboxylic acids and create new opportunities for integrating these biocatalysts into CoA-dependent biosynthetic pathways relevant to chemical biomanufacturing and synthetic biology.