Zou Yutong, Bailey Constance B
School of Chemistry, University of Sydney, Sydney, NSW Australia.
Biophys Rev. 2024 Sep 26;16(5):591-603. doi: 10.1007/s12551-024-01234-1. eCollection 2024 Oct.
Cell-free protein synthesis (CFPS) has emerged as an attractive platform for biotechnology and synthetic biology due to its numerous advantages to cell-based technologies for specific applications. CFPS can be faster, less sensitive to metabolite toxicity, and amenable to systems that are not easily genetically manipulated. Due to these advantages, a promising application of CFPS is to characterize biosynthetic gene clusters, particularly those harbored within the genomes of microorganisms that generate secondary metabolites, otherwise known as natural products. In the postgenomic era, genome sequencing has revealed an incredible wealth of metabolic diversity. However, far more of these pathways are termed "cryptic," i.e., unable to be produced under standard laboratory conditions than have been characterized. A major barrier to characterizing these cryptic natural products using CFPS is that many of these pathways require utilization of complex cofactors, many of which to date are not recycled efficiently or in an economically viable fashion. In this perspective, we outline strategies to regenerate cofactors relevant to secondary metabolite production in CFPS. This includes adenosine 5'-triphosphate, coenzyme A, redox cofactors (iron-sulfur clusters, nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide), all of which play a crucial role in important biosynthetic enzymes. Such advances in cofactor recycling enable continuous production of complex metabolites in CFPS and expand the utility of this emergent platform.
无细胞蛋白质合成（CFPS）已成为生物技术和合成生物学中一个颇具吸引力的平台，因为相较于基于细胞的技术在特定应用中具有诸多优势。CFPS可以更快，对代谢物毒性不那么敏感，并且适用于不易进行基因操作的系统。由于这些优势，CFPS的一个有前景的应用是对生物合成基因簇进行表征，特别是那些存在于产生次级代谢产物（即天然产物）的微生物基因组中的基因簇。在后基因组时代，基因组测序揭示了令人难以置信的丰富代谢多样性。然而，与已被表征的相比，这些途径中更多被称为“隐秘的”，即在标准实验室条件下无法产生。使用CFPS表征这些隐秘天然产物的一个主要障碍是，这些途径中的许多需要利用复杂的辅因子，其中许多到目前为止都没有以有效或经济可行的方式进行循环利用。从这个角度来看，我们概述了在CFPS中再生与次级代谢产物生产相关辅因子的策略。这包括腺苷5'-三磷酸、辅酶A、氧化还原辅因子（铁硫簇、烟酰胺腺嘌呤二核苷酸磷酸、黄素腺嘌呤二核苷酸），所有这些在重要的生物合成酶中都起着关键作用。辅因子循环利用方面的这些进展能够在CFPS中持续生产复杂代谢产物，并扩展这个新兴平台的效用。
