Synthesis of myrcene using a hybrid cell-free reaction system

Myrcene is a typical monoterpene with applications as pharmaceutical product, biofuel, and so forth. However, only traces of myrcene accumulate in plants, and in vivo production of monoterpene in microbial cells also contains multiple restrictive factors. To alleviate this limitation, a hybrid cell-free metabolic engineering (CFME) system was developed in this study and a series of CFME strategies were employed for the engineering of myrcene synthase from glucose. First, a convenient CFME system was established, in which Pichia pastoris and Escherichia coli lysates enriched with MVA pathway and myrcene synthase respectively were used as “plug and play” modules and combined. After optimization, the myrcene titer was significantly improved by 119.26%. Second, rational design of myrcene synthase was performed by conserved residues modification and substrate tunnel engineering. Myrcene synthase variant N238S outperformed the wild-type enzyme in CFME system, and the myrcene titer value reached 56.80 mg/L. Molecular dynamics simulations provided significant mechanistic insights into the enhanced catalytic capability and myrcene production. This work highlights the feasibility of hybrid CFME system to connect tandem module and sustain complex metabolism for myrcene biomanufacturer. It also illustrates the value of the cell-free approach as a starting point for the rational engineering of monoterpene synthase.