Designing an enzyme assembly line for green cascade processes using bio-orthogonal chemistry

Two non-canonical amino acids (ncAAs) with bio-orthogonal reactive groups, namely, p-azido-l-phenylalanine (p-AzF) and p-propargyloxy-l-phenylalanine (p-PaF), were genetically inserted into an aldo-keto reductase (AKR) and an alcohol dehydrogenase (ADH), respectively, at two preselected sites for each enzyme. The variants were expressed in the genome recoded bacterium Escherichia coli C321.ΔA. Supernatants of the individual cell lysates were subsequently mixed to produce orderly combi-crosslinked enzymes (O-CLEs) of AKR and ADH by co-polymerization of the two variants through their reactive bio-orthogonal groups. The site-specific cross-linked enzymes (S-CLEs) and cross-linked enzyme aggregates (CLEAs) were produced using dibenzocycloocta-4a,6a-diene-5,11-diyne (DBA) and glutaraldehyde as the crosslinking agent, respectively. The catalytic efficiencies of the O-CLEs, S-CLEs and combi-CLEAs were determined using the water soluble dihydro-4, 4-dimethyl-2, 3-furandione as a surrogate substrate in aqueous solution at 37 °C. The O-CLEs exhibited the highest catalytic efficiency (K_cat/K_M = 11.36 S⁻¹ mM⁻¹) that was 4.24 and 22.27 times that of S-CLEs and combi-CLEAs, respectively. In the asymmetric cascade synthesis of (R)-1-(2-chlorophenyl) ethanol the product yield after 14 h using the O-CLEs, S-CLEs and the combi-CLEAs was 93%, 55% and 16%, respectively. Moreover, high activities and selectivity (ee > 99.99%) were maintained at high substrate concentrations in prolonged operation.