Genetically engineered host bacteria have an extensive history for the production of specific proteins including the synthesis of single enzymes for the modification of compounds produced for industrial purposes by biological or chemical processes. Such processes have been developed largely through the process of discovery. The ability to assemble multiple enzymes into synthetic pathways is a new development aided by the synthetic biology approach of constructing and assembling suitable enzymes into pathways that may or may not occur in Nature to provide a high-impact platform for bio-manufacturing of chemicals, biofuels and pharmaceuticals. Industry has depended on chemical catalysts because of the known constraints experienced frequently with biological catalysts but when high stereochemistry, mild synthesis conditions and environmentally friendly processes are significant, application of enzymes is preferred, especially in the case of drug synthesis where enantioselectivity is important. However, many whole cell production processes are beset by toxicity problems, metabolite competition, the production of side products, sub-optimal enzyme ratios and varying temperature optima. As a result, a cell-free biocatalysis allows the manipulation of substrate ratios, provision of regenerated cofactors and adjustment of high energy flux ratios that are difficult or impossible to control in whole cell synthesis. We discuss here the construction of cell free biocatalytic pathways as added free enzymes or multi-enzyme modules that may contain heterologous catalysts. We examine the status of applications leading to commercialisation, emphasizing the importance of economical cofactor regeneration. Nevertheless, problems remain, particularly protein post-translational modifications including glycosylation, phosphorylation, ubiquitination, acetylation, proteolysis, etc. The National Renewable Energy Laboratory and Pacific North West Laboratory have published lists of top value-added chemicals from biomass that include glucaric acid. There have been few reports on the combination of synthetic biology and cell-free protein synthesis to set up pathways for these valuable intermediate compounds. This observation is despite the existence of at least one large scale but specialized cell-free production of antibody conjugates. This review will provide a description of one successful attempt at the cell-free production of glucaric acid and will evaluate progress for other key intermediate and platform chemicals.

基因改造的宿主细菌在生产特定蛋白质方面具有广泛的历史，包括合成单一酶以修饰通过生物学或化学过程工业化生产的化合物。这些过程很大程度上是通过发现过程开发的。将多种酶组装到合成途径中的能力是一种新的发展，它是通过将合适的酶构建和组装到自然界中可能出现或可能不存在的途径中的合成生物学方法而得到的，从而为化学品，生物燃料的生物制造提供了高影响力的平台和药品。由于生物催化剂经常遇到的已知限制，因此工业一直依赖化学催化剂，但是当立体化学高度复杂时，温和的合成条件和对环境友好的工艺非常重要，尤其是在对映选择性很重要的药物合成情况下，最好使用酶。然而，许多全细胞生产过程都受到毒性问题，代谢物竞争，副产物的生产，次优酶比例和变化的温度最佳状态的困扰。结果，无细胞的生物催化作用允许操纵底物比例，提供再生的辅因子以及调节在整个细胞合成中难以或不可能控制的高能量通量比例。我们在这里讨论无细胞生物催化途径的构建，将其作为可能包含异源催化剂的添加的游离酶或多酶模块。我们研究了导致商品化的应用程序的状态，强调经济的辅因子再生的重要性。尽管如此，仍然存在问题，特别是蛋白质的翻译后修饰，包括糖基化，磷酸化，泛素化，乙酰化，蛋白水解等。美国国家可再生能源实验室和西北太平洋实验室已经发布了生物量包括高加索酸的高附加值化学品清单。关于合成生物学和无细胞蛋白质合成相结合以建立这些有价值的中间体化合物的途径的报道很少。尽管存在至少一种大规模但专门的无细胞的抗体偶联物生产，但该观察结果仍然存在。