Artificial in Vitro Synthetic Enzymatic Biosystem for the One-Pot Sustainable Biomanufacturing of Glucosamine from Starch and Inorganic Ammonia

Glucosamine is a naturally occurring bioactive amino monosaccharide which is widely used in the food, cosmetics, and pharmaceutical industries. It is traditionally produced by acid hydrolysis/enzymolysis of chitin but suffers from possible individual allergenic risk, serious environmental pollution, and low production yield. A recent rising approach for glucosamine production is microbial fermentation using engineered microorganisms to manufacture N-acetylglucosamine, followed by acid hydrolysis of N-acetylglucosamine to glucosamine. However, this approach does not produce glucosamine in one-pot, and suffers from environmental issues caused by the acid hydrolysis process. To produce cost-competitive glucosamine with environmental sustainability, we demonstrated an artificial in vitro synthetic enzymatic biosystem comprised of five cascade enzymes for biomanufacturing glucosamine from maltodextrin and inorganic ammonia in one-pot. This nonshellfish-derived, phosphate-balanced, and coenzyme-free synthetic enzymatic biosystem was optimized in terms of pH value, enzyme composition, phosphate concentration, and ammonia concentration. The optimized enzyme cocktail converted 10 g/L maltodextrin to 7.9 g/L glucosamine with a conversion efficiency of 75.8% mol/mol glucose equivalent of maltodextrin. Moreover, a simple scaling-up for this nonfermentative biomanufacturing biosystem was performed to investigate its industrial potential, giving out 23.7 g/L glucosamine from 50 g/L maltodextrin. This in vitro enzymatic platform provides a biorefinery technology for the production of glucosamine featuring predictable security, environmental friendliness, and sustainability, thereby representing a highly promising alternative approach for glucosamine production on an industrial scale.