Bober & Nair (2019) Nature Communications D-Tagatose is a rare ketohexose sugar with sweetness similar to that of sucrose. However, its glycemic index and caloric value is much lower because of low bioavailability, making it an attractive GRAS (generally regarded as safe) sugar substitute. Recent studies have also demonstrated that it is anti-hyperglycemic and prebiotic, which promotes gut health. The enzyme L-arabinose isomerase (LAI) that isomerizes L-arabinose to L-ribulose can also isomerize D-galactose to D-tagatose. A variety of LAIs from different microorganisms have been isolated and have reported optimal activity at a range of temperatures and pH. Some of the limitations of tagatose biosynthesis using LAI that may be hindering commercial viability are, 1) unfavorable enzymatic kinetics since galactose is not the native substrate of LAI, 2) low enzyme stability, and 3) low equilibrium constant for galactose to tagatose isomerization. Few previous reports have been successful at engineering enzymatic properties of LAI for industrial application; often addressing only one of the bottlenecks to productivity. To address the kinetic issue, several groups have used enzyme engineering methods to enhance activtity of LAI toward galactose. To counter low-stability issues, many groups have tested the utility of thermophilic enzymes. However, high reaction temperatures (≥ 80 °C) result in significant caramelization, which is undesirable. Finally, the thermodynamic limitations of isomerization arguably, the most recalcitrant issue since ΔG°rxn ≈ +1.2 kcal/mol. There have currently been no studies that look to systematically analyze all three limitations – kinetic, thermodynamic, and enzyme stability. This work clearly demonstrates the presence of these three limitations and provides a novel approach to balance their advantages and limitations. We engineered the probiotic bacterium Lactobacillus plantarum as expression host in this approach that enabled ~50 % conversion of galactose to tagatose in 4 h (productivity of ~38 mmol tagatose L-1 h-1) ultimately reaching ~85 % conversion after 48 h at high galactose loading (300 mM) in batch culture. This is among the highest conversions and productivities reported to date for tagatose production using a mesophilic enzyme. Such an approach is expected to be applicable to other biocatalytic systems where similar trade-offs between kinetics, thermodynamics, and/or stability pose hurdles to process development.

Surpassing thermodynamic, kinetic, and stability barriers to biocatalytic isomerization of galactose to tagatose

ACS Fall 2020 Virtual Meeting

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