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
. 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.
Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, ...alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.
Synthetic biochemistry, the cell-free production of biologically based chemicals, is a potentially high-yield, flexible alternative to in vivo metabolic engineering. To limit costs, cell-free systems ...must be designed to operate continuously with minimal addition of feedstock chemicals. We describe a robust, efficient synthetic glucose breakdown pathway and implement it for the production of bioplastic. The system's performance suggests that synthetic biochemistry has the potential to become a viable industrial alternative.
Synthetic biochemistry seeks to engineer complex metabolic pathways for chemical conversions outside the constraints of the cell. Establishment of effective and flexible cell-free systems requires ...the development of simple systems to replace the intricate regulatory mechanisms that exist in cells for maintaining high-energy cofactor balance. Here we describe a simple rheostat that regulates ATP levels by controlling the flow down either an ATP-generating or non-ATP-generating pathway according to the free-phosphate concentration. We implemented this concept for the production of isobutanol from glucose. The rheostat maintains adequate ATP concentrations even in the presence of ATPase contamination. The final system including the rheostat produced 24.1 ± 1.8 g/L of isobutanol from glucose in 91% theoretical yield with an initial productivity of 1.3 g/L/h. The molecular rheostat concept can be used in the design of continuously operating, self-sustaining synthetic biochemistry systems.
Metabolic engineering efforts that harness living organisms to produce natural products and other useful chemicals face inherent difficulties because the maintenance of life processes often runs ...counter to our desire to maximize important production metrics. These challenges are particularly problematic for commodity chemical manufacturing where cost is critical. A cell-free approach, where biochemical pathways are built by mixing desired enzyme activities outside of cells, can obviate problems associated with cell-based methods. Yet supplanting cell-based methods of chemical production will require the creation of self-sustaining, continuously operating systems where input biomass is converted into desired products at high yields, productivities, and titers. We call the field of designing and implementing reliable and efficient enzyme systems that replace cellular metabolism, synthetic biochemistry.
Cost-effective production of chemicals in engineered organisms is challenging because there is a basic conflict between the needs of the cell for life, and our needs for commercial viability.The synthetic biochemistry approach can free us from cellular limitations, opening up new possibilities for enhancing biobased chemical production parameters.Cost-effective cell-free bioreactor construction requires designing simple, robust systems that can run efficiently for long periods of time. While considerable challenges remain before biomanufacturing is possible at the scale currently used in microbial systems, recent progress suggests that a cell-free approach has promise.
Abstract
Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to ...achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L
−1
h
−1
, a titer of 275 g L
−1
and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.
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
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.
Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25-45°C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the ...Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca²⁺ coordination may explain how these lipases can fold without specific chaperones.
The greatest potential environmental benefit of metabolic engineering would be the production of high-volume commodity chemicals, such as biofuels. Yet, the high yields required for the economic ...viability of low-value chemicals is particularly hard to achieve in microbes owing to the myriad competing biochemical pathways. An alternative approach, which we call synthetic biochemistry, is to eliminate the organism by constructing biochemical pathways in vitro. Viable synthetic biochemistry, however, will require simple methods to replace the cellular circuitry that maintains cofactor balance. Here we design a simple purge valve module for maintaining NADP(+)/NADPH balance. We test the purge valve in the production of polyhydroxybutyryl bioplastic and isoprene--pathways where cofactor generation and utilization are unbalanced. We find that the regulatory system is highly robust to variations in cofactor levels and readily transportable. The molecular purge valve provides a step towards developing continuously operating, sustainable synthetic biochemistry systems.
The high yields required for the economical production of chemicals and fuels using microbes can be difficult to achieve due to the complexities of cellular metabolism. An alternative to performing ...biochemical transformations in microbes is to build biochemical pathways in vitro, an approach we call synthetic biochemistry. Here we test whether the full mevalonate pathway can be reconstituted in vitro and used to produce the commodity chemical isoprene. We construct an in vitro synthetic biochemical pathway that uses the carbon and ATP produced from the glycolysis intermediate phosphoenolpyruvate to run the mevalonate pathway. The system involves 12 enzymes to perform the complex transformation, while providing and balancing the ATP, NADPH, and acetyl‐CoA cofactors. The optimized system produces isoprene from phosphoenolpyruvate in ∼100% molar yield. Thus, by inserting the isoprene pathway into previously developed glycolysis modules it may be possible to produce isoprene and other acetyl‐CoA derived isoprenoids from glucose in vitro.
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