Modular Whole-Cell Transaminase Cascades for Bioamination of Biomass-Derived Aldehydes through Amino-Donor Regeneration and Pyruvate Removal

Abstract

The biocatalytic conversion of lignocellulose-derived aldehydes into value-added aromatic and heteroaromatic amines is often restricted by the reversibility of transamination, inefficient amine-donor utilization, pyruvate accumulation, and insufficient intracellular cofactor regeneration. In this study, three modular whole-cell cascade systems were constructed by coexpressing the ω-transaminase RtTA with alanine dehydrogenase (AlaDH), lactate dehydrogenase and formate dehydrogenase (LDH–FDH), or pyruvate decarboxylase (PDC). The RtTA–AlaDH module was designed to regenerate L-alanine from pyruvate and ammonium using reducing equivalents supplied by endogenous glucose metabolism. The RtTA–LDH–FDH system coupled pyruvate reduction with formate-dependent NADH regeneration, whereas the RtTA–PDC cascade irreversibly converted pyruvate to acetaldehyde and CO2. Vanillin was used as the model substrate to compare the effects of reaction temperature, pH, donor or cosubstrate loading, organic-solvent content, and substrate concentration. Among the three systems, the RtTA–PDC cascade showed the best performance at elevated vanillin loading, producing 67.2 mM vanillylamine from 75 mM vanillin in approximately 90% yield at an L-alanine-to-vanillin molar ratio of 7:1. The RtTA–LDH–FDH system maintained transamination activity over a broader range of vanillin concentrations than the RtTA–AlaDH cascade, whereas the latter avoided direct supplementation with L-alanine. The engineered whole-cell systems also catalyzed the transamination of isovanillin, p-anisaldehyde, furfural, and 5-hydroxymethylfurfural and retained catalytic activity in aqueous media containing dimethyl sulfoxide. These results highlight auxiliary-enzyme selection as a modular strategy for tuning whole-cell transamination performance.