Asymmetric C–C bond formation mediated by aldolase provides one of the most efficient ways to produce valuable chemicals in biomanufacturing. Dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GA3P) are two important platform compounds for asymmetric C–C bond formation. In this study, several artificial ATP-free in vitro synthetic enzymatic biosystems were constructed to produce valuable chemicals via facile synthesis of GA3P and DHAP from starch and pyrophosphate. Six cascade enzymes were used for the biotransformation of starch and pyrophosphate to GA3P or DHAP: alpha-glucan phosphorylase (αGP), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), pyrophosphate phosphofructokinase (PPi-PFK), d-fructose 1,6-bisphosphate aldolase (FruA), and triosephosphate isomerase (TIM). These two compounds were then used to produce various chemicals, including 2-deoxy-d-ribose (DR) and rare ketoses. After the optimization of reaction conditions, ∼23.2 mM DR with a product yield of 96.7% and 15.2 mM d-allulose with a product yield of 95.0% were produced, both achieving near-stoichiometric yields through downstream aldol additions and dephosphorylation reactions in one pot. In addition, more than 80% of the product yields of DR and many rare ketoses, such as d-allulose, l-tagatose, d-sorbose, l-fructose, and d-xylulose, from high concentrations of substrates were obtained, showing high industrial potential. This in vitro biomanufacturing platform may provide a promising and cost-effective approach for biomanufacturing value-added chemicals through asymmetric C–C bond formation in the near future.

中文翻译：

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人工合成的无ATP体外合成酶生物系统促进醛缩酶介导的C–C键形成以进行生物制造

由醛缩酶介导的不对称C–C键形成提供了在生物制造中生产有价值的化学物质的最有效方法之一。磷酸二羟基丙酮酯（DHAP）和3-磷酸d-甘油醛（GA3P）是形成不对称C–C键的两个重要平台化合物。在这项研究中，构建了几个人工合成的无ATP的体外合成酶生物系统，可以通过从淀粉和焦磷酸盐轻松合成GA3P和DHAP来生产有价值的化学物质。六个级联酶用于将淀粉和焦磷酸生物转化为GA3P或DHAP：α-葡聚糖磷酸化酶（αGP），磷酸葡萄糖变位酶（PGM），磷酸葡萄糖异构酶（PGI），焦磷酸磷酸果糖激酶（PPi-PFK），d-果糖1,6-二磷酸醛缩酶（FruA）和磷酸三糖异构酶（TIM）。然后这两种化合物被用于生产各种化学品，包括2-脱氧d -核糖（DR）和稀有酮糖。在优化反应条件之后，产生了约23.2 mM的DR，产物产率为96.7％和15.2 mM d-阿洛糖，产物产率为95.0％，通过添加下游的羟醛和在一锅中进行脱磷酸反应，均达到了接近化学计量的产率。 。此外，DR和许多稀有的酮糖，例如d-阿洛糖，l-塔格糖，d-山梨糖，l-果糖和d的产品收率超过80％从高浓度的底物中获得了β-木酮糖，显示出很高的工业潜力。这个体外生物制造平台可能会在不久的将来通过不对称的C–C键形成为生物制造增值化学品提供一种有前途且具有成本效益的方法。