Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades**

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof-of-principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross-reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O-methylnorbelladine

Development of Continuous Flow Systems to Access Secondary Amines Through Previously Incompatible Biocatalytic Cascades

From Wiley via Jisc Publications RouterHistory: received 2021-03-17, rev-recd 2021-04-12, pub-electronic 2021-05-19Article version: VoRPublication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/ L013762/1, BB/M027791/1, BB/M02903411, BB/ M028836/1Funder: H2020 European Research Council; Grant(s): 788231-ProgrES-ERC-2017-ADGAbstract: A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out multistep cascades in a controlled and selective way. As biocatalytic cascades get more complex, reactions become unattainable under typical batch conditions. Here a number of continuous flow systems were used to overcome batch incompatibility, thus allowing for successful biocatalytic cascades. As proof‐of‐principle, reactive carbonyl intermediates were generated in situ using alcohol oxidases, then passed directly to a series of packed‐bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase/transaminase/imine reductase sequence, introducing different amine reagents at each step without cross‐reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product 4O‐methylnorbelladine

Rapid Model-Based Optimization of a Two-Enzyme System for Continuous Reductive Amination in Flow

Enzymes are increasingly combined into multienzyme systems for cost and productivity benefits. Further advantages can be gained through the use of immobilized enzymes, allowing continuous biotransformations in flow. However, the optimization of such multienzyme systems is challenging, particularly where immobilized enzymes are used. Here, we meet this challenge using both mechanistic and empirical modeling to optimize a reaction involving a reductive aminase and a glucose dehydrogenase for continuous biocatalytic reductive amination in flow. Crucially, the construction of the mechanistic model was achieved quickly, with only a few important parameters determined experimentally, and ensemble modeling used to facilitate the use of estimates or literature values. Upon reaching the limits of the mechanistic model’s capabilities, we show that solution space can be further explored using a definitive screening design to generate an empirical model of the reaction, using the mechanistic model’s prediction as a starting point. We demonstrate the use of this empirical model to design optimal processes for either high productivity or to minimize necessary cofactor and cosubstrate concentrations. Our results demonstrate that the synergistic use of both mechanistic and empirical modeling offers a route for rapid optimization of multienzyme systems of immobilized enzymes in flow with minimal experimental effort

Production of High Value Amine Intermediates via Biocatalytic Cascades in Continuous Flow

A key aim of biocatalysis is to mimic the ability of eukaryotic cells to carry out compartmentalized multistep cascades in a controlled and selective way. As biocatalytic cascades get longer and more complex, reactions become unattainable under typical batch conditions. Here a continuous flow multipoint injection reactor was combined with switching valves to overcome batch incompatibility, thus allowing for successful biocatalytic reaction cascades. As proof-of-principle, several reactive carbonyl intermediates were generated in situ using galactose oxidase and engineered choline oxidases, then passed directly to a series of packed-bed modules containing different aminating biocatalysts which accordingly produced a range of structurally distinct amines. The method was expanded to employ a batch incompatible sequential amination cascade via an oxidase-transaminase-imine reductase sequence, introducing different amine reagents at each step without cross reactivity. The combined approaches allowed for the biocatalytic synthesis of the natural product alkaloid precursor 4O-methylnorbelladine. The flow biocatalysis platform shown here significantly increases the scope of novel biocatalytic cascades, removing previous limitations due to reaction and reagent batch incompatibility.</jats:p