Cell-free enzymatic reaction cascades combine the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. The absence of a regulatory cell environment enables direct process control including methods for facile bottleneck identification and process optimization. Within this work, we developed a reduced, enzymatic reaction cascade for the direct production of L-alanine from D-glucose and ammonium sulfate. An efficient, activity based enzyme selection is demonstrated for the two branches of the cascade. The resulting redox neutral cascade is composed of a glucose dehydrogenase, two dihydroxyacid dehydratases, a keto-deoxy-aldolase, an aldehyde dehydrogenase and an L-alanine dehydrogenase. This artificial combination of purified biocatalysts eliminates the need for phosphorylation and only requires NAD as cofactor. We provide insight into in detail optimization of the process parameters applying a fluorescamine based L-alanine quantification assay. An optimized enzyme ratio and the necessary enzyme load were identified and together with the optimal concentrations of cofactor (NAD), ammonium and buffer yields of >95% for the main branch and of 8% for the side branch were achieved.

The aldehyde dehydrogenase from Thermoplasma acidophilum was previously implemented as a key enzyme in a synthetic cellfree reaction cascade for the production of alcohols. In order to engineer the enzyme's cofactor specificity from NADP + to NAD + , we identified selectivity-determining residues with the CSR-SALAD tool and investigated further positions based on the crystal structure. Stepwise combination of the initially discovered six point mutations allowed us to monitor the cross effects of each mutation, resulting in a final variant with reduced K M for the non-native cofactor NAD + (from 18 to 0.6 mM) and an increased activity for the desired substrate D-glyceraldehyde (from 0.4 to 1.5 U/mg). Saturation mutagenesis of the residues at the entrance of the substrate pocket could eliminate substrate inhibition. Molecular dynamics simulations showed a significant gain of flexibility at the cofactor binding site for the final variant. The concomitant increase in stability against isobutanol and only a minor reduction in its temperature stability render the final variant a promising candidate for future optimization of our synthetic cell-free enzymatic cascade.