Enhanced Production of Pinene by Using a Cell-Free System with Modular Cocatalysis

α-Pinene is an important monoterpene that is widely used as a pharmaceutical product, biofuel, and so forth. We first established a cell-free system with modular cocatalysis for the production of pinene from glucose. After optimization of the compositions of the cell-free reaction mixture using the Plackett-Burman experimental design and the path of steepest ascent, the production of pinene increased by 57%. It was found that ammonium acetate, NAD⁺, and NADPH are the three most important parameters for the production of pinene. Mix-and-match experiments showed that the simultaneous addition of the lysate of Escherichia coli overexpressing native 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, SufBCD Fe-S cluster assembly protein, isopentenyl-diphosphate isomerase, and Pinus taeda pinene synthase improved the production of pinene. Increasing the enzyme concentration of the extract further enhanced the production of pinene to 1256.31 ± 46.12 mg/L with a productivity of 104.7 mg/L h, almost 1.2-fold faster than any system reported thus far. This study demonstrates that a cell-free system is a powerful and robust platform for biomanufacture.