Myo-Inositol (vitamin B8) is widely used in the drug, cosmetic, and food & feed industries. Here, we present an in vitro non-fermentative enzymatic pathway that converts starch to inositol in one vessel. This in vitro pathway is comprised of four enzymes that operate without ATP or NAD supplementation. All enzyme BioBricks are carefully selected from hyperthermophilic microorganisms, that is, alpha-glucan phosphorylase from Thermotoga maritima, phosphoglucomutase from Thermococcus kodakarensis, inositol 1-phosphate synthase from Archaeoglobus fulgidus, and inositol monophosphatase from T. maritima. They were expressed efficiently in high-density fermentation of Escherichia coli BL21(DE3) and easily purified by heat treatment. The four-enzyme pathway supplemented with two other hyperthermophilic enzymes (i.e., 4-α-glucanotransferase from Thermococcus litoralis and isoamylase from Sulfolobus tokodaii) converts branched or linear starch to inositol, accomplishing a very high product yield of 98.9 ± 1.8% wt./wt. This in vitro (aeration-free) biomanufacturing has been successfully operated on 20,000-L reactors. Less costly inositol would be widely added in heath food, low-end soft drink, and animal feed, and may be converted to other value-added biochemicals (e.g., glucarate). This biochemical is the first product manufactured by the in vitro synthetic biology platform on an industrial scale. Biotechnol. Bioeng. 2017;114: 1855-1864. © 2017 Wiley Periodicals, Inc.

Cell-free synthetic enzymatic biosystem is emerging to expand the traditional biotechnological mode by utilizing a number of purified/partially purified enzymes and coenzymes in a single reaction vessel for the production of desired products from low-cost substrates. Here, a cell-free synthetic biosystem containing minimized number of reactions was designed for the conversion of d-glucose to l-lactate via pyruvate. This NADH-balanced biosystem was comprised of only 5 thermophilic enzymes without ATP supplementation. After optimization of enzyme loading amounts, buffer concentration and cofactor concentration, d-glucose was converted to l-lactate with a product yield of ∼90%. Our study has provided an emerging platform with potentials in producing pyruvate-derived chemicals, and may promote the development of cell-free synthetic enzymatic biosystems for biomanufacturing.

Disaccharides are valuable oligosaccharides with an increasing demand in the food, cosmetic, and pharmaceutical industries. Disaccharides can be manufactured by extraction from the acid hydrolysate of plant-derived substrates, but this method has several issues, such as the difficulty in accessing natural substrates, laborious product separation processes, and troublesome wastewater treatment. A chemical synthesis using glucose was developed for producing disaccharides, but this approach suffers from a low product yield due to the low specificity and requires tedious protection and deprotection processes. In this study, we adopted an artificial strategy for producing a variety of value-added disaccharides from low-cost starch through the construction of an in vitro synthetic enzymatic platform: two enzymes worked in parallel to convert starch to glucose and glucose 1-phosphate, and these two intermediates were subsequently condensed together to a disaccharide by a disaccharide phosphorylase. Several disaccharides, such as laminaribiose, cellobiose, trehalose, and sophorose, were produced successfully from starch with the yields of more than 80% with the help of kinetic mathematical models to predict the optimal reaction conditions, exhibiting great potential in an industrial scale. This study provided a promising alternative to reform the mode of disaccharide manufacturing.