Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades.

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.

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

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.

ATP-Independent and Cell-Free Biosynthesis of ß-Hydroxy Acids Using Vinyl Esters as Smart Substrates.

In vitro biosynthetic pathways that condense and reduce molecules through coenzyme A (CoASH) activation demand energy and redox power in the form of ATP and NAD(P)H, respectively. These coenzymes must be orthogonally recycled by ancillary reactions that consume chemicals, electricity, or light, impacting the atom economy and/or the energy consumption of the biosystem. In this work, we have exploited vinyl esters as dual acyl and electron donor substrates to synthesize ß-hydroxy acids through a non-decarboxylating Claisen condensation, reduction and hydrolysis stepwise cascade, including a NADH recycling step, catalyzed by a total of 4 enzymes. Herein, the chemical energy to activate the acyl group with CoASH and the redox power for the reduction are embedded into the vinyl esters. Upon optimization, this self-sustaining cascade reached a titer of (S)-3-hydroxy butyrate of 24 mM without requiring ATP and simultaneously recycling CoASH and NADH. This work illustrates the potential of in vitro biocatalysis to transform simple molecules into multi-functional ones.