Construction of A Filamentous Self-assembling Protein Scaffold-mediated Dual-enzyme Catalytic Cascade System for Enhanced D-Tagatose Synthesis
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Abstract:
D-tagatose is a rare hexose that is weakly metabolized but highly sweet. It is thus broadly useful in the food industry. Most current biosyntheses of D-tagatose utilize wild type and modified versions of the key enzyme L-arabinose isomerase. The use of a multi-enzyme catalytic cascade to synthesize D-tagatose remains under-explored, while existing methods often exhibit a poor conversion rate due to thermodynamic equilibrium constraints. In this study, we constructed and characterized a filamentous self-assembled protein scaffold EE/KK derived from Methanocaldococcus jannaschii. This scaffold facilitates efficient cascade interactions between fluorescent proteins both intracellularly and extracellularly. Using this scaffold, D-xylose reductase (SsXR, an NAD(P)H-dependent D-xylose reductase derived from Scheffersomyces stipitis) and galactitol dehydrogenase (RlGDH, an SDR family oxidoreductase derived from Rhizobium leguminosarum) were assembled in Escherichia coli BL21(DE3). This approach significantly enhanced the efficiency of D-tagatose synthesis through the oxidoreductase pathway. D-tagatose yield in the EE/KK cascade system increased by 50% relative to that from the free-fraction system. Further optimization of fermentation conditions in the recombinant strain BL21-EX/KG (where EX and KG denote the protein complexes EE-SsXR and KK-RlGDH, respectively) revealed that using Luria-Bertani (LB) medium at 20 ℃, with 0.1 mmol/L isopropyl-β-D-thiogalactopyranoside and 10 g/L lactose as substrate yielded 3.93 g/L D-tagatose, corresponding to a lactose conversion rate of 0.39 g/g, or 74% of the theoretical complete conversion rate (0.53 g/g), outperforming most reports of D-tagatose synthesis using lactose as a substrate. This research introduces a promising E. coli strain for efficient D-tagatose biosynthesis, and an effective tool for assembly of multi-enzyme catalytic cascades.
