J Agric Food Chem. 2019 Dec 11;67(49):13430-13436. doi: 10.1021/acs.jafc.9b00413. Epub 2019 Apr 8.
Naringenin is an important precursor for the production of a wide spectrum of flavonoids, and its production is of great interest in metabolic engineering. However, in cellular systems, identification of rate-limiting factors is often difficult because of complex regulatory networks. Cell-free catalytic systems emerge as a promising method to address this issue. Here, we explored the cell-free biosystem for naringenin production by combining different sources of 4-coumaroyl-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). After systematic analysis of enzyme levels, substrate concentrations, and cofactors, 4CL and CHS were found to be crucial to the reaction. The best loading ratio of 4CL/CHS/CHI was 10:10:1, and malonyl-CoA was the limiting factor, as identified previously in fermentation. For the first time, we successfully constructed the system for naringenin production . Our study will deepen our understanding of the key factors in naringenin production and guide further engineering.
柚皮苷是产生广泛类黄酮的重要前体物质，其生产在代谢工程中具有重要意义。然而，在细胞系统中，由于复杂的调控网络，确定限速因素往往很困难。无细胞催化系统作为一种有前途的方法出现，以解决这个问题。在这里，我们通过结合不同来源的 4-香豆酰辅酶 A 连接酶（4CL）、查尔酮合酶（CHS）和查尔酮异构酶（CHI），探索了柚皮苷生产的无细胞生物系统。经过对酶水平、底物浓度和辅助因子的系统分析，发现 4CL 和 CHS 对反应至关重要。4CL/CHS/CHI 的最佳加载比为 10:10:1，正如之前在发酵中确定的那样，丙二酰辅酶 A 是限制因素。我们首次成功构建了柚皮苷生产系统。我们的研究将加深我们对柚皮苷生产关键因素的理解，并指导进一步的工程设计。
