A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.
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机译：描述了一种新的合成平台，该平台具有生产几种稀有糖的潜力，以L-核糖为模型目标。合成构建了编码来自芹菜（花园芹菜）的独特的NAD依赖性甘露醇-1-脱氢酶（MDH）的基因，以便在大肠杆菌中最佳表达。这种MDH酶催化几种多元醇及其左旋糖对应物的相互转化，包括核糖醇向左旋核糖的转化。成功地以活性形式表达了重组MDH，并证明了一步纯化。使用创建的重组大肠杆菌菌株作为全细胞催化剂，证明了其可用于生产L-核糖的合成工具，并使用摇瓶实验对系统进行了改进。已确定添加50至500μMZnCl2和添加5 g / L甘油均可提高产量。在40 g /升的浓度下，最终的转化率> 70％，在100 g /升的浓度下> 50％。然后将确定的最佳条件扩大到1升发酵，在72小时内导致100克/升核糖醇的55％转化，容积生产率为17.4克升 -1 天 −1 。该系统代表了大规模生产1-核糖的显着改进的方法。
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