丝状自组装蛋白支架介导的双酶级联催化体系构建促进D-塔格糖合成

Construction of A Filamentous Self-assembling Protein Scaffold-mediated Dual-enzyme Catalytic Cascade System for Enhanced D-Tagatose Synthesis

D-塔格糖是一种稀有的己酮糖,具有低能量、高甜度,在食品领域具有广泛的应用。目前,D-塔格糖的生物合成大多聚焦于异构酶途径及关键酶L-阿拉伯糖异构酶的挖掘与改造,多酶级联催化的应用较少,且受热力学平衡影响转化率较低。该研究首先构建并表征了来源于Methanocaldococcus jannaschii的丝状自组装蛋白支架EE/KK,结果显示,其在胞内外均可实现有效的荧光蛋白级联互作。其次,在大肠杆菌Escherichia coli BL21(DE3)中应用EE/KK蛋白支架级联组装D-木糖还原酶(SsXR,Scheffersomyces stipitis来源的NAD(P)H-dependent D-xylose Reductase)和半乳糖醇脱氢酶(RlGDH,Rhizobium leguminosarum来源的SDR Family Oxidoreductase),强化基于氧化还原酶途径合成D-塔格糖的效率。相较于游离体系,EE/KK级联体系的D-塔格糖产量提高50%。进一步对级联体系重组菌株BL21-EX/KG(EX和KG分别为EE-SsXR和KK-RlGDH蛋白复合体的缩写)发酵条件进行优化,结果表明:以Luria-Bertani(LB)培养基为发酵培养基,温度20℃,0.1 mmol/L异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-Thiogalactopyranoside,IPTG)下,以10 g/L乳糖为底物,D-塔格糖产量可达3.93 g/L,对乳糖转化率为0.39 g/g,为理论转化率(0.53 g/g)的74%,高于大多数以乳糖为底物合成D-塔格糖的报道。该研究为D-塔格糖生物合成提供潜在的高效菌株以及多酶级联组装提供有效的工具。

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.