Bioproduction of D-Tagatose in Escherichia coli by Harnessing the Reverse Leloir Pathway

Abstract

D-Tagatose is a natural, rare monosaccharide containing 38% of the calories of sucrose. As an alternative sweetener, it offers numerous benefits including a low glycemic index, prebiotic properties, and the ability to lower blood sugar levels. Currently, tagatose is largely produced by industrial isomerization of galactose using both chemical and enzymatic catalysis by a class of enzymes called L-arabinose isomerases (LAIs). While these processes are effective, they are laborious and use expensive feedstocks. This study proposes and characterizes a novel, fully in vivo pathway to D-tagatose from a minimal-cost feedstock, glucose, using an Escherichia coli microbial bioproduction process. This process is possible due to the discovery and characterization of a phosphatase from, Dictyostelium discoideum, which exhibits selective dephosphorylation of galactose-1-phosphate to make free galactose using E.coli cells. By expressing this phosphatase, along with an LAI in a modified strain background that eliminates the cell’s galactose catabolism potential, this system demonstrates the ability to make tagatose from glucose. The highest yields of tagatose from this project are currently 5.4%. However, it is also demonstrated that the system can be further optimized to achieve up to 77% increases in galactose, and 23% increases in tagatose. Additionally, this project shows unprecedented yields of galactose made from glucose in vivo (31.5%). Showing the potential of a novel system such as this demonstrates the attainability of a cheap, simple, and efficient process to make rare sugars. This could not only lead to healthier products but ones more broadly accessible to the public.