Two non-canonical amino acids (ncAAs) with bio-orthogonal reactive teams, specifically, p-azido-L-phenylalanine (p-AzF) and p-propargyloxy-L-phenylalanine (p-PaF), have been genetically inserted into an aldo-keto reductase (AKR) and an alcohol dehydrogenase (ADH), respectively, at two preselected websites for every enzyme. The variants have been expressed within the genome recoded bacterium Escherichia coli C321.ΔA. Supernatants of the person cell lysates have been subsequently blended to supply orderly combi-crosslinked enzymes (O-CLEs) of AKR and ADH by co-polymerization of the 2 variants via their reactive bio-orthogonal teams. The location-specific cross-linked enzymes (S-CLEs) and cross-linked enzyme aggregates (CLEAs) have been produced utilizing dibenzocycloocta-4a,6a-diene-5,11-diyne (DBA) and glutaraldehyde because the crosslinking agent, respectively. The catalytic efficiencies of the O-CLEs, S-CLEs and combi-CLEAs have been decided utilizing the water soluble dihydro-4, 4-dimethyl-2, 3-furandione as a surrogate substrate in aqueous answer at 37 °C. The O-CLEs exhibited the best catalytic effectivity (Okay_cat/Okay_M = 11.36 S⁻¹ mM⁻¹) that was 4.24 and 22.27 occasions that of S-CLEs and combi-CLEAs, respectively. Within the uneven cascade synthesis of (R)-1-(2-chlorophenyl) ethanol the product yield after 14 h utilizing the O-CLEs, S-CLEs and the combi-CLEAs was 93%, 55% and 16%, respectively. Furthermore, excessive actions and selectivity (ee > 99.99%) have been maintained at excessive substrate concentrations in extended operation.