A bio-inspired cell-free system for cannabinoid production from inexpensive inputs.

Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l−1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production. A cell-free system for cannabinoid production uses only low-cost inputs with 12 enzymes and can operate either aerobically or anaerobically, in addition to reducing ATP requirements by use of an engineered system for malonate-CoA biosynthesis.