The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

The increasing demand for chiral compounds supports the development of enzymatic processes. Dehydrogenases are often the enzymes of choice due to their high enantioselectivity combined with broad substrate acceptance. However, their requirement on costly NAD­(P)/H as cofactor has sparked interest in the development of biomimetic derivatives that are easy to synthesize and, therefore, less expensive. Until now, few reactions with biomimetics have been described and regeneration is limited to nonenzymatic means, which are not suitable for incorporation and in situ approaches. Herein, we describe a regeneration enzyme, glucose dehydrogenase from Sulfolobus solfataricus (SsGDH), and demonstrate its activity with different biomimetics with the structure nicotinamide ring-alkyl chain-phenyl ring. Subsequent enzyme engineering resulted in the double mutant SsGDH Ile192Thr/Val306Ile, which had a 10-fold higher activity with one of the biomimetics compared with the wild-type enzyme. Using this engineered variant in combination with an enoate reductase from Thermus scotoductus resulted in the first enzyme-coupled regeneration process for biomimetic cofactor without ribonucleotide or ribonucleotide analogue and full conversion of 10 mM 2-methylbut-2-enal with 1-phenethyl-1,4-dihydropyridine-3-carboxamide as cofactor.

α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation. The five enzymes (including one for cofactor recycling) were first investigated individually to define optimal reaction conditions for the cascade reaction. Then, the kinetic parameters were determined under these conditions and the inhibitory effects of substrate, intermediates, and product were evaluated. As cofactor supply is critical for the cascade reaction, various set-ups were tested: increasing concentrations of the recycling enzyme, different initial NAD concentrations, as well as the use of a bubble reactor for faster oxygen diffusion. Finally, we were able to convert 10gL glucuronate with 92% yield of aKG within 5h. The maximum productivity of 2.8gL h is the second highest reported in the biotechnological synthesis of aKG.

In analyzing the reductive power of Escherichia coli K-12 for metabolic engineering approaches, we identified YahK and YjgB, two medium-chain dehydrogenases/reductases subgrouped to the cinnamyl alcohol dehydrogenase family, as being important. Identification was achieved using a stepwise purification protocol starting with crude extract. For exact characterization, the genes were cloned into pET28a vector and expressed with N-terminal His tag. Substrate specificity studies revealed that a large variety of aldehydes but no ketones are converted by both enzymes. YahK and and YjgB strongly preferred NADPH as cofactor. The structure of YjgB was modeled using YahK as template for a comparison of the active center giving a first insight to the different substrate preferences. The enzyme activity for YahK, YjgB, and YqhD was determined on the basis of the temperature. YahK showed a constant increase in activity until 60 °C, whereas YjgB was most active between 37 and 50 °C. YqhD achieved the highest activity at 50 °C. Comparing YjgB and Yahk referring to the catalytic efficiency, YjgB achieved for almost all substrates higher rates (butyraldehyde 221 s−1 mM−1, benzaldehyde 1,305 s−1 mM−1). Exceptions are the two substrates glyceraldehydes (no activity for YjgB) and isobutyraldehyde (YjgB 0.26 s−1 mM−1) which are more efficiently converted by YahK (glyceraldehyde 2.8 s−1 mM−1, isobutyraldehyde 14.6 s−1 mM−1). YahK and even more so YjgB are good candidates for the reduction of aldehydes in metabolic engineering approaches and could replace the currently used YqhD.

Oxidoreductases are attractive biocatalysts that convert achiral substrates into products of higher value, but they are also for the most part dependent on nicotinamide cofactors. Recently, biomimetic nicotinamide derivatives have received attention as less costly alternatives to natural cofactors. However, recycling of biomimetics is still challenging because there are only limited opportunities. Here, we have characterized various biomimetic cofactors with regard to stability and redox potentials to find the best alternative to natural cofactors. Further, the cofactor spectrum of NADH oxidase from Lactobacillus pentosus (LpNox) could be expanded, and the enzymatic activity was also compared to activities with different small-molecule catalysts. As a result, we succeeded in identifying several strategies for regeneration of oxidized biomimetics.

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