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 in vitro. Our study will deepen our understanding of the key factors in naringenin production and guide further engineering.
Keywords: biocatalysis; cell-free; flavonoid; in vitro biosynthesis; naringenin.