Development of isopentenol utilization pathway based on cell-free metabolic engineering and its application in multienzyme cascades synthesis.

Conventional microbial biosynthetic pathways for isoprenoid often require intricate metabolic balancing and multigene regulation, which are not only time-consuming but also prone to interference from host cellular metabolism and feedback inhibition by toxic intermediate metabolites or end-products. In this study, we developed a novel cell-free multienzyme catalytic system for the synthesis of lycopene, a representative isoprenoid. By integrating the isopentenol utilization pathway with cell-free metabolic engineering strategies, the system eliminates the need for complex in vivo genetic modifications and avoids inherent cellular metabolic interference. The in vitro reaction employs seven enzymes directly, enabling rapid and efficient lycopene production without relying on costly substrates isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The optimized cell-free reaction mixture consisted of isoprenol and prenol (3:1 molar ratio, 15mM), seven key enzymes (each at a concentration of 0.6mg/mL), 8mM Mg2+, 1mM NAD, 1mM ATP, 1mM CoA, and 0.05% (w/v) Tween 20 in 30mM Bis-Tris buffer (pH 6.5). After incubation at 30 °C for 20hours, the lycopene titer reached 128.04mg/L. Further in vivo validation achieved a final lycopene titer of 270.0mg/L. This novel approach offers a promising alternative strategy to overcome common challenges associated with the construction and regulation of complex natural isoprenoid biosynthetic pathways.