Cell-free enzymatic production of mannitol from fructose

Overview

This solution offers a cell-free enzymatic process for producing mannitol, a naturally occurring sweetener with excellent thermal and pH stability. Mannitol remains stable at low pH (2.7–3.2) and ambient temperatures, and can withstand heat shocks of 200–250 °C for 30 seconds, making it attractive for food, pharmaceutical, and industrial applications. The process converts fructose—a sugar readily sourced from sucrose or cellulose depolymerization—into mannitol using purified, immobilized mannitol dehydrogenase (MDH). By operating without living cells, the platform sidesteps common fermentation challenges such as byproduct formation, microbial contamination, and complex downstream separation.

Technical specifications

Core approach:

• Recombinant expression of NADH-dependent mannitol dehydrogenase in Generally Regarded As Safe (GRAS) organisms
• Enzyme purification and immobilization for reuse and improved process stability
• Cell-free reaction system using fructose-rich substrates such as high fructose corn syrup
• NADH cofactor regeneration to drive high-yield conversion

Enzyme sourcing strategy:

• Ten phylogenetically diverse mannitol dehydrogenases will be cloned and expressed in the yeast Pichia pastoris, selected for its GRAS status and capacity for secreted enzyme production
• Each candidate enzyme will be evaluated for biochemical properties including stability and turnover number
• The most efficient MDH will be engineered for direct secretion into fermentation broth, eliminating the need for cell lysis
• Scale-up will be performed in a fermentor to support industrial production volumes

Prior validation: The lab has demonstrated related cell-free systems producing isobutanol from alpha-ketovaleric acid at yields approaching 90% using a three-enzyme cascade with NADH cofactor regeneration, establishing the feasibility of the underlying cell-free metabolic engineering framework.

Technology readiness level

The technology is currently at an early-to-mid stage of development. The underlying cell-free metabolic engineering approach—combining enzyme cofactor regeneration, enzyme scaffolding, and multi-enzyme cascades—has been validated with a related three-enzyme system achieving near-90% yields. The mannitol-specific platform is now advancing through enzyme screening and expression optimization in Pichia pastoris, followed by immobilization testing and scale-up fermentation. Next steps include evaluating ten candidate MDH enzymes for stability and catalytic efficiency, engineering secretion of the best-performing variant, and demonstrating high-yield mannitol production from corn syrup at fermentor scale.