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bioRxiv · 10.1101/2024.12.16.628640

Efficient microbial glucose-fructose conversion at ambient temperature through adaptive laboratory evolution and application for D-allulose production

Lehnert, A.·Gentile, R.·Tahiraj, C.·Wirtz, A.·Baumgart, M.·Polen, T.·Gohlke, H.·Bott, M.
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Abstract

The low-calorie sugar D-allulose is a promising alternative to D-sucrose and high-fructose corn syrup, but its microbial production from D-glucose at mesophilic temperatures is limited by insufficient D-glucose isomerase (XylA) activity. Here, we overcome this bottleneck by evolving a Corynebacterium glutamicum selection strain whose growth strictly depends on XylA function. This strategy yielded a XylA variant with a nine-fold higher catalytic efficiency, sugar transporter variants (IolT1) with ten-fold increased activity for D-glucose and D-fructose, and hints for co-transport of these sugars by the D-sucrose transporter PtsS. Molecular dynamics simulations provided possible mechanistic explanations for the adaptive mutations. Combining the evolved enzymes with a suitable D-allulose 3-epimerase in a highly engineered chassis strain enabled whole-cell conversion of D-glucose to D-allulose with a 15% yield at 30 {degrees}C. This performance rivals immobilized enzyme processes performed at [~]60{degrees}C while avoiding enzyme purification and immobilization, offering an alternative for low-calorie sweetener production.

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Lehnert, A., Gentile, R., Tahiraj, C., Wirtz, A., Baumgart, M., Polen, T., Gohlke, H., Bott, M.. 2024-12-19. Efficient microbial glucose-fructose conversion at ambient temperature through adaptive laboratory evolution and application for D-allulose production. https://doi.org/10.1101/2024.12.16.628640

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