Multistep Synthesis of UDP-Glucose Using Tailored, Permeabilized Cells of E. coli

Abstract

We constructed and applied a recombinant, permeabilized Escherichia coli strain for the multistep synthesis of UDP-glucose. Sucrose phosphorylase (E.C. 2.4.1.7) of Leuconostoc mesenteroides was over expressed and the pgm gene encoding for phosphoglucomutase (E.C. 5.4.2.2) was deleted in E. coli to yield the E. coli JW 0675-1 SP strain. The cells were permeabilized with the detergent Triton X-100 at 0.05 % v/v. The synthesis of UDP-glucose with permeabilized cells was then optimized with regard to pH, cell density during the synthesis and growth phase during cell harvest, metal cofactor, other media components, and temperature. In one configuration sucrose, phosphate, UMP, and ATP were used as substrates. At pH 7.8, 27 mg/ml cell dry weight, cell harvest during the early stationary phase of growth and Mn(2+) as cofactor a yield of 37 % with respect to UMP was achieved at 33 °C. In a second step, ATP was regenerated by feeding glucose and using only catalytic amounts of ATP and NAD(+). A UDP-glucose yield of 60 % with respect to UMP was obtained using this setup. With the same setup but without addition of external ATP, the yield was 54%.

Diversity of phosphorylases in glycoside hydrolase families

“…The concomitant actions of galactokinase and 1, 3-β-galactosyl-N-acetylhexosamine phosphorylase were used in the chemoenzymatic syntheses of the derivatives of lacto-N-tetraose (Yao et al 2015) and derivatives of GNB (Li et al 2015). UDP-Glc was prepared using engineered E. coli cells expressing sucrose phosphorylase as the catalysts (Weyler and Heinzle 2015). De Bruyn et al (2015) introduced the sucrose phosphorylase gene in E. coli to increase fermenting production of phenolic glucosides using glucosyltransferase and enhancing the intracellular pool of UDP-Glc.…”

Role of phosphoglucomutase in regulating trehalose metabolism in Nilaparvata lugens

“…G-6-P is a central, metabolite, part of glycolysis and the pentose phosphate pathway, providing precursors for anabolic pathways and cofactors required for cell proliferation (Ward and Thompson 2012). PGM is widely found in animals, plants, and microorganisms and is distributed in almost all tissues (Egli et al 2010;Stray-Pedersen et al 2014;Weyler and Heinzle 2015). In the brown planthopper, both PGM1 and PGM2 were most highly expressed in the midgut, followed by the foot (Fig.…”

“…PGM is a conserved enzyme, ubiquitous in animals, plants, and microorganisms catalysing the interconversion of glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P) (Jin et al 2018;Egli et al 2010;Stray-Pedersen et al 2014;Weyler and Heinzle 2015;Liu 2013). Because glucose-6-phosphate is an important central metabolite, PGM plays an important role in the metabolism of proteins, lipids, and nucleic acids and is key for the development of plants (Egli et al 2010;Paparelli et al 2013;Malinova et al 2014) and some microorganisms (Liu 2013).…”

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

“…A major advantage of working with living cells is their ability to conveniently regenerate expensive cofactors like ATP with their native metabolic pathways. In one study, the overexpression of LmSP allowed permeabilised E. coli to efficiently produce UDP-glucose from sucrose and UMP by generating a steady intracellular pool of Glc1P as intermediate, while deletion of the gene coding for phosphoglucomutase prevented this pool from being depleted via glycolysis [41].…”