Coenzyme A Thioester Intermediates as Platform Molecules in Cell‐Free Chemical Biomanufacturing

Abstract

The in vitro synthesis of Coenzyme A (CoA)‐thioester intermediates opens new avenues to transform simple molecules into more complex and multifunctional ones by assembling cell‐free biosynthetic cascades. In this review, we have systematically cataloged known CoA‐dependent enzyme reactions that have been successfully implemented in vitro. To faciliate their identification, we provide their UniProt ID when available. Based on this catalog, we have organized enzymes into three modules: activation, modification, and removal. i) The activation module includes enzymes capable of fusing CoA with organic molecules. ii) The modification module includes enzymes capable of catalyzing chemical modifications in the structure of acyl‐CoA intermediates. And iii) the removal module includes enzymes able to remove the CoA and release an organic molecule different from the one activated in the upstream. Based on these reactions, we constructed a reaction network that summarizes the most relevant CoA‐dependent biosynthetic pathways reported until today. From the information available in the articles, we have plotted the total turnover number of CoA as a function of the product titer, observing a positive correlation between both parameters. Therefore, the success of a CoA‐dependent in vitro pathway depends on its ability to regenerate CoA, but also to regenerate other cofactors such as NAD(P)H and ATP.

Chemo-enzymatic Synthesis of Coenzyme A Using Copurified Enzymes from Probiotic Escherichia coli Nissle

“…Coenzyme A (CoA) engages in numerous biochemical reactions with the functional acyl-thioester groups . As a crucial cofactor throughout three domains of life, CoA regulates cellular energy metabolism by modulating substrate/product concentrations, enzyme activity, and gene expression through the acetylation/deacetylation of histone and transcription factor .…”

“…In contrast, in vitro biotransformation is a thriving platform that relieves constraints in microbial fermentation, such as cell duplication, metabolic flux balance, and energy requirements . Several in vitro CoA-dependent biosynthetic pathways have been reported, where CoA is involved in the reactions of activation, modification, and removal to synthesize more complex molecules such as terpenes, polyketides, and polyhydroxyalkanoates . Therefore, an efficient and sustainable platform for in vitro CoA manufacturing is necessary, offering advantages by introducing only the required enzymes, minimizing side reactions and potentially yielding a higher CoA titer.…”

“…Coenzyme A (CoA) engages in numerous biochemical reactions with the functional acyl-thioester groups. 1 As a crucial cofactor throughout three domains of life, CoA regulates cellular energy metabolism by modulating substrate/product concentrations, enzyme activity, and gene expression through the acetylation/deacetylation of histone and transcription factor. 2 The significance of CoA extends to the antioxidant response by the low-molecular-weight thiols group, which is accumulated during the presence of excess reactive oxygen species.…”

“…13 Several in vitro CoA-dependent biosynthetic pathways have been reported, where CoA is involved in the reactions of activation, modification, and removal to synthesize more complex molecules such as terpenes, polyketides, and polyhydroxyalkanoates. 14 Therefore, an efficient and sustainable platform for in vitro CoA manufacturing is necessary, offering advantages by introducing only the required enzymes, minimizing side reactions and potentially yielding a higher CoA titer. However, in vitro CoA production includes multiple ATP-dependent reactions, requiring an excessive amount of expensive ATP (∼$100/g) for efficient CoA production.…”