Abstract：
We designed and constructed a green and sustainable bioprocess to efficiently coproduce D -tagatose, bioethanol, and microbial protein from whey powder. First, a one-pot biosynthesis process involving lactose hydrolysis and D -galactose redox reactions for D -tagatose production was established in vitro via a three-enzyme cascade. Second, a nicotinamide adenine dinucleotide phosphate-dependent galactitol dehydrogenase mutant, D36A/I37R, based on the nicotinamide adenine dinucleotide-dependent polyol dehydrogenase from Paracoccus denitrificans was created through rational design and screening. Moreover, an NADPH recycling module was created in the oxidoreductive pathway, and the tagatose yield increased by 3.35-fold compared with that achieved through the pathway without the cofactor cycle. The reaction process was accelerated using an enzyme assembly with a glycine-serine linker, and the tagatose production rate was 9.28-fold higher than the initial yield. Finally, Saccharomyces cerevisiae was introduced into the reaction solution, and 266.5 g of D -tagatose, 162.6 g of bioethanol, and 215.4 g of dry yeast (including 38% protein) were obtained from 1 kg of whey powder (including 810 g lactose). This study provides a promising sustainable process for functional food (D -tagatose) production. Moreover, this process fully utilized whey powder, demonstrating good atom economy.
摘要：
我们设计并构建了一个绿色和可持续的生物过程，以有效地共同生产D-塔格糖，生物乙醇，和乳清粉中的微生物蛋白。首先,通过三酶级联反应，在体外建立了涉及乳糖水解和D-半乳糖氧化还原反应以生产D-塔格糖的一锅式生物合成过程。第二,烟酰胺腺嘌呤二核苷酸磷酸依赖性半乳糖醇脱氢酶突变体，D36A/I37R,基于烟酰胺腺嘌呤二核苷酸依赖性多元醇脱氢酶从反硝化副球菌通过合理的设计和筛选创建。此外，在氧化还原途径中创建了一个NADPH回收模块，与没有辅因子循环的途径相比，塔格糖的产量增加了3.35倍。使用具有甘氨酸-丝氨酸接头的酶装配加速反应过程,塔格糖的生产率比初始产量高9.28倍。最后,将酿酒酵母引入反应溶液中，和266.5克D-塔格糖，162.6克生物乙醇，从1kg乳清粉(包括810g乳糖)中获得215.4g干酵母(包括38%蛋白质)。这项研究为功能性食品（D-塔格糖）生产提供了一个有前途的可持续过程。此外，这个过程充分利用乳清粉，展示了良好的原子经济性。
