‘Tiny factories’: bioengineered E. coli produce rare, low-calorie sugar substitute

Scientists have bioengineered the bacterium Escherichia coli to produce tagatose – a rare, low-calorie alternative to table sugar.

A fresh breakthrough in the search for natural sweeteners has seen researchers from Tufts University, Manus Bio (both MA, USA) and Kcat Enzymatic Private Limited (Bengaluru, India), who have successfully engineered E. coli to convert glucose directly into the sugar substitute tagatose. The development could bypass long-standing stumbling blocks in tagatose biosynthesis and prove advantageous for the sustainable production of other rare sugars.

D-Tagatose (tagatose) is a natural sweetener comparable in sweetness to sucrose but with a fraction of the calories. It is ‘generally recognized as safe’ by the Food and Drug Administration, making it an attractive sugar replacement for use in a variety of consumer products. However, due to its scarcity in nature, tagatose is typically manufactured, rather than extracted, and current approaches for doing so are inefficient and expensive, relying on isomerization of the pricey and less plentiful sugar galactose.

It is in this landscape that the team designed a biosynthetic route to tagatose production, introducing a whole-cell process in E. coli that converts glucose directly to tagatose by reversing the Leloir pathway.

The Leloir pathway is the primary route for native galactose metabolism in E. coli and other organisms, whereby galactose is converted into glucose-1-phosphate for use in glycolysis and central metabolism. To reverse this process, the researchers engineered the bacteria to produce Gal1P phosphatase (Gal1Pase) – a newly discovered enzyme from the slime mold Dictyostelium discoideum. Doing so provided the thermodynamic driving force to shift the pathway in the opposite direction, enabling glucose-to-galactose conversion as the first step in a tagatose biosynthetic pathway.

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Computational analyses of Gal1Pase revealed hydrogen bond networks that explain how the enzyme so stringently discriminates its substrate to achieve this.

By co-expressing Gal1Pase with a second enzyme called L-arabinose isomerase in a metabolically engineered strain, the team demonstrated direct conversion of glucose to tagatose.

“We developed a way to produce tagatose by engineering the bacteria Escherichia coli to work as tiny factories, loaded with the right enzymes to process abundant amounts of glucose into tagatose,” explained study author Nik Nair. “This is much more economically feasible than our previous approach.”

Based on the researchers’ estimations, this pathway could theoretically yield up to 94.9% tagatose – compared to just 50% for traditional strategies that isomerize galactose produced from lactose.

This may only be a proof-of-principle study, and further optimization is required, but it’s a promising development that could see tagatose’s star rise as a healthier alternative to sucrose. And its potential doesn’t end there.

“Beyond its immediate application to tagatose, our pathway provides a generalizable framework for the biosynthesis of galactose-derived molecules directly from glucose,” the researchers write in their conclusion, adding that it’s “a new starting point for future engineering of rare sugar pathways.”