Molecular Dynamics Analysis of a Rationally Designed Aldehyde Dehydrogenase Gives Insights into Improved Activity for the Non-Native Cofactor NAD.

The aldehyde dehydrogenase from was previously implemented as a key enzyme in a synthetic cell-free 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 for the non-native cofactor NAD (from 18 to 0.

Optimization of a reduced enzymatic reaction cascade for the production of L-alanine.

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.