多酶级联系统制备蒜糖醇的细胞工厂构建及工艺优化

Construction and Process Optimization of Cell Factory for Allitol Synthesis with Multienzymatic Cascade System

摘要:

蒜糖醇是一种在食品、医药等领域具有较大应用前景的新型功能性甜味剂。为实现蒜糖醇的绿色生物合成,基于稀有糖转化策略通过共表达葡萄糖异构酶、D-阿洛酮糖3-差向异构酶及核糖醇脱氢酶,在大肠杆菌体内建立了利用廉价底物D-葡萄糖生产蒜糖醇的多酶级联系统；并通过偶联甲酸脱氢酶构建辅因子循环系统,提高胞内还原型辅酶Ⅰ的再生效率。为平衡多酶级联系统中的个体表达差异,以全细胞催化活性为指标,对细胞工厂的发酵条件和催化条件进行了优化。结果表明:重组菌在20℃、1.00mmol/L异丙基-β-D-硫代半乳糖苷的发酵条件下,诱导24h可得到较佳的蛋白质表达量；在40℃、50mmol/L Tris-HCl (pH值8.0)及5.0g/L甲酸钠的催化条件下,全细胞可在15h内转化25.00g/L的D-葡萄糖生成19.33g/L蒜糖醇。通过表达系统的优化进一步降低了底物和中间产物的残留量,蒜糖醇的产量提高至21.12g/L。研究旨在为全细胞生物转化法实现蒜糖醇的规模化合成提供理论依据,探索以预处理甘蔗糖蜜为底物生产蒜糖醇的可行性,希望对延长糖产业链,提高糖产品附加值起到积极作用。

Abstract:

Allitol is generally used as a novel functional sweetener with great potential applications in food, medical care and other fields. Based on the Izumoring strategy, a multienzymatic cascade system involved glucose isomerase, D-psicose 3-epimerase and ribitol dehydrogenase was established in Escherichia coli for the production of allitol from cheap substrate D-glucose. The cofactor recycling system was constructed using the formate dehydrogenase to improve the regeneration efficiency of intracellular reduced nicotinamide adenine dinucleotide. To balance the individual expression differences in the multienzymatic cascade system, the fermentation and catalytic conditions of the cell factory were optimized using whole-cell catalytic activity as an indicator. According to the results, the recombinant bacteria could obtain better protein expression was obtained when cells were incubated at 20℃ for 24h with 1.00mmol/L isopropyl-β-D-thiogalactoside. Under catalytic conditions of 40℃ and 50mmol/L Tris-HCl (pH 8.0) with 5.0g/L sodium formate, the recombinant cells could produce 19.33g/L allitol from 25.00g/L D-glucose. The residual amounts of substrate and intermediates were significantly reduced and the yield of allitol was further improved to 21.12g/L through expression system optimization. The whole-cell biotransformation method could provide a theoretical basis for the large-scale synthesis of allitol, accompany with producing allitol from pretreated sugarcane molasses, which could have positive significance in extending the sugar industry chain and increasing the added value of sugar products.