Abstract
Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We obtain conversion yields >95% and titres >15 g l−1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.
Highlights
Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells
Our results suggest that commodity chemicals and pharmaceutical intermediates derived from acetyl-CoA and ATP are accessible through synthetic biochemistry systems
Our results show that industrially relevant complex biomolecules such as monoterpenes can be produced in vitro directly from glucose using a synthetic biochemistry platform
Summary
Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. The titres are an order of magnitude over cellular toxicity limits and difficult to achieve using cell-based systems Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals. In the case of monoterpenes limonene and pinene, a cellular toxicity limit of r0.5% (5 g l À 1) is a major contributor to low titres and presents a key barrier for production in a microbial host[27,28]. Cell-free production provides an alternative approach to chemical transformations that can ease the technical challenges of engineering microorganisms and the limitations imposed by requiring cell viability[26]. Our results suggest that commodity chemicals and pharmaceutical intermediates derived from acetyl-CoA and ATP are accessible through synthetic biochemistry systems
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