An in vitro synthetic biology platform for the industrial biomanufacturing of myo‐inositol from starch

Abstract

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.

Stoichiometric Regeneration of ATP by A NAD(P)/CoA‐free and Phosphate‐balanced In Vitro Synthetic Enzymatic Biosystem

“…After 24 h of reaction at 37 °C, the residual maltodextrin was 5 mM in both systems (Figure a), indicating 4.6 mM maltodextrin was metabolized. The incomplete utilization of maltodextrin was due to the incapability of αGP to phosphorolyze the branched chains of maltodextrin . In the reaction system without the CR module, 1 molecule of ATP (and hence 1 molecule of L‐theanine) would be regenerated from 1 glucose equivalent of maltodextrin.…”

“…The incomplete utilization of maltodextrin was due to the incapability of αGP to phosphorolyze the branched chains of maltodextrin. [22] In the reaction system without the CR module, 1 molecule of ATP (and hence 1 molecule of L-theanine) would be regenerated from 1 glucose equivalent of maltodextrin. As expected, 4.6 mM of L-theanine was produced finally (Figure 2a and Figure S3).…”

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In Vitro Hydrogen Production at Elevated Temperature

“…[24] The strains E. coli BL21(DE3) containing the protein expression plasmids of mG6PDH-RfDoc, DI-CcDoc, DRT-L-mCtDoc and miniscaffoldin were cultivated in LB medium supplemented with 1.2 %g lycerol at 37 8C. [24] The strains E. coli BL21(DE3) containing the protein expression plasmids of mG6PDH-RfDoc, DI-CcDoc, DRT-L-mCtDoc and miniscaffoldin were cultivated in LB medium supplemented with 1.2 %g lycerol at 37 8C.…”

Extending Designed Linear Biocatalytic Cascades for Organic Synthesis

“…The conversion of starch to value‐added myo‐inositol was reported through an in vitro non‐fermentative five‐enzyme pathway (Scheme ) . This co‐factor independent pathway comprised five enzymes that operate without ATP or NAD + supplementation.…”