The quest to achieve a sustainable supply of both energy and chemicals is one of the great challenges of this century. 5-(Hydroxymethyl)furfural (HMF), the long-known dehydration product of hexose carbohydrates, has become an important nexus for access to both liquid fuels and chemicals. One such biofuel is 2,5-dimethylfuran (DMF), which is a product of HMF hydrogenolysis and contains an energy density 40% greater than that of ethanol. In recent years, much work has been done to effect the chemical conversion of fructose, glucose, cellulose, and even lignocellulosic biomass into HMF in high yield. Here, we provide an overview of methods to access HMF from carbohydrates with the highest potential to reach an industrial scale, along with a discussion of unmet technological needs necessary for commercialization.
The primary objective of this work was to create a cell-free protein synthesis extract that produces proteins requiring disulfide bonds while using glucose as an energy source. We attempted to avoid using iodoacetamide (IAM) to stabilize the required oxidizing thiol redox potential, since previous IAM pretreatments prevented glucose utilization apparently by inactivating glyceraldehyde 3-phosphate dehydrogenase (G-3PDH). Instead, the glutathione reductase (Gor)-mediated disulfide reductase system was disabled by deleting the gor gene from the KC6 cell-extract source strain. The thioredoxin reductase (TrxB)-mediated system was disabled by first adding a purification tag to the trxB gene in the chromosome to create strain KGK10 and then by affinity removal of the tagged TrxB. This was expected to result in a cell extract devoid of all disulfide reductase activity, but this was not the case. Although the concentration of IAM required to stabilize oxidized glutathione in the KGK10 extract could be reduced 20-fold, IAM pretreatment was still required to avoid disulfide reduction. Nonetheless, active urokinase and murine granulocyte macrophage-colony stimulating factor (mGM-CSF) were produced in reactions with KGK10 extract either with affinity removal of TrxB or with 50 microM IAM pretreatment. With the less intensive IAM pretreatment, glucose could be used as an energy source in a production system that promotes oxidative protein folding. This new protocol offers an economically feasible cell-free system for the production of secreted mammalian proteins as human therapeutics or vaccines.
Escherichia coli thioredoxin reductase (TR) and glutathione reductase (GR) are dimeric proteins that require a £avin adenine dinucleotide (FAD) cofactor for activity. A cellfree protein synthesis (CFPS) reaction supplemented with FAD was used to produce TR at 760 W Wg/ml with 89% of the protein being soluble. GR accumulated to 521 W Wg/ml in a cell-free reaction with 71% solubility. The TR produced was fully active with a speci¢c activity of 1390 min 31 . The GR had a speci¢c activity of 139 U/mg, which is signi¢cantly more active than reported for GR puri¢ed from cells. The speci¢c activity for both TR and GR decreased without FAD supplementation. This research demonstrates that CFPS can be used to produce enzymes that are multimeric and require a cofactor. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
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