Workflow for Biocatalytic Reaction Performance Prediction and Analysis

The development of predictive tools to assess enzyme mutant performance and physical organic approaches to enzyme mechanistic interrogation are crucial to the field of biocatalysis. While many indispensable tools exist to address qualitative aspects of biocatalytic reaction design, they often require extensive experimental data sets or a priori knowledge of reaction mechanism. However, quantitative prediction of enzyme performance is lacking. Herein, we present a workflow that merges both computational and experimental data to produce statistical models that predict the performance of new substrates and enzyme mutants while also providing insight into reaction mechanism. As a validating case study, this platform was applied to investigate a non-native enantioselective photoenzymatic radical cyclization. Statistical models enabled interrogation of the reaction mechanism, and the predictive capabilities of these same models led to the quantitative prediction of the enantioselectivities of new substrates with several enzyme mutants. This platform was constructed for application to any biocatalytic system wherein mechanistic interrogation, prediction of reaction performance with new substrates, or quantitative performance of enzyme mutants would be desirable. Overall, this proof of concept study provides a new tool to complement existing protein engineering and reaction design strategies.

Developing an Ethanol Utilization Pathway based NADH Regeneration System in Escherichia coli

Interests remain in searching for cofactor regeneration system with higher efficiency at lower substrate cost. Glucose dehydrogenase (GDH) system has been dominant in NADH regeneration, but it only has a theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-step ethanol utilization pathway (EUP) in pathway-based NADH regeneration. The pathway runs from ethanol to acetaldehyde and to acetyl-CoA with each step generating one NADH, that together results in a higher theoretical yield of two NADH per ethanol molecule. In this project, anaerobic biotransformation of ketone (acetophenone or butanone) to alcohol by cpsADH from Candida parapsilosis was used as readout for evaluating relative efficacy and operating modes for EUP cofactor regeneration in Escherichia coli BL21 (DE3). Experiment tests validated that EUP was more efficient than GDH in NADH regeneration. Further, growing cell delivered higher biotransformation efficiency compared to resting cell due to the driving force generated by cell growth. Finally, preculture or cultivation in M9 + 10 g/L ethanol medium delivered higher biotransformation efficiency compared to LB medium. Overall, EUP could help regenerate NADH in support of a biocatalytic reaction, and is more efficient in cofactor regeneration than GDH.

Production of bio-xylitol from d-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

Background Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from d-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from d-xylose. Results Pichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform d-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield. Conclusions We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform d-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of d-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

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

<div> <p>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 <i>in situ </i>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 <i>via </i>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.</p> </div> <b><br></b>

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

<div> <p>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 <i>in situ </i>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 <i>via </i>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.</p> </div> <b><br></b>

Diastereoselective Synthesis of b-Branched a-Amino Acids via Bio-catalytic Dynamic Kinetic Resolution

We report a biocatalytic transamination method to prepare a broad range of b-branched a-amino acids that proceeds with high diastereo- and enantioselectivity. Mechanistic studies show that the transformation proceeds through a dynamic kinetic resolution process that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction is applied to the synthesis of several cyclic fragments and in the first total synthesis of jomthonic acid A.

Diastereoselective Synthesis of b-Branched a-Amino Acids via Bio-catalytic Dynamic Kinetic Resolution

We report a biocatalytic transamination method to prepare a broad range of b-branched a-amino acids that proceeds with high diastereo- and enantioselectivity. Mechanistic studies show that the transformation proceeds through a dynamic kinetic resolution process that is unique to the optimal enzyme. To highlight its utility and practicality, the biocatalytic reaction is applied to the synthesis of several cyclic fragments and in the first total synthesis of jomthonic acid A.

Surface Permeability of Membrane and Catalytic Performance Based on Redox-Responsive of Hybrid Hollow Polymeric Microcapsules

“Smart” polymeric microcapsules with excellent permeability of membranes have drawn considerable attention in scientific and industrial research such as drug delivery carriers, microreactors, and artificial organelles. In this work, hybrid hollow polymeric microcapsules (HPs) containing redox-active gold-sulfide bond were prepared with bovine serum albumin, inorganic metal cluster (AuNCs), and poly(N-isopropylacrylamide) conjugates by using Pickering emulsion method. HPs were transferred from water-in-oil to water-in-water by adding PEGbis(N-succinimidylsuccinate). To achieve redox-responsive membrane, the Au-S bond units incorporated into the microcapsules’ membranes, allowed us to explore the effects of a new stimuli, that is, the redox Au-S bond breaking on the microcapsules’ membranes. The permeability of these hybrid hollow polymeric microcapsules could be sensitively tuned via adding environment-friendly hydrogen peroxide (H2O2), resulting from a fast fracture of Au-S bond. Meanwhile, AuNCs and conjugates could depart from the microcapsules, and enhance the permeability of the membrane. Based on the excellent permeability of the membrane, phosphatase was encapsuled into HPs and p-nitrophenyl phosphate as a substrate. After adding 1 × 10−2 and 1 × 10−4 M H2O2, the catalytic efficiency was nearly 4.06 and 2.22 times higher than that of HPs in the absence of H2O2, respectively. Hence, the unique redox-responsive HPs have potential applications in biocatalytic reaction, drug delivery, and materials as well as in bioscience.

Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

Background: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose.Results: P. pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.Conclusions: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.