Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an [alpha]-Gal epitope

• Published in: Microbial cell factories Vol. 9; p. 1
• Main Authors: Ruffing, Anne M, Chen, Rachel R
• Format: Journal Article
• Language: English
• Published: London BioMed Central Ltd 12.01.2010; BioMed Central
• Subjects: Agrobacterium tumefaciens; Analysis; Antigenic determinants; Bacteria; E coli; Enzymes; Genetic aspects; Glucose; Lactose; Metabolic engineering; Microbiology; Monkeys & apes; Sucrose; Vaccines; Varieties; United States
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/1475-2859-9-1

Oligosaccharides containing a terminal Gal-[alpha]1,3-Gal moiety are collectively known as [alpha]-Gal epitopes. [alpha]-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the [alpha]-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the [alpha]-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the [alpha]-Gal epitope, Gal-[alpha]1,3-Lac. Agrobacterium sp. ATCC 31749 was engineered to produce Gal-[alpha]1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:[alpha]1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the [alpha]-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting [alpha]-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-[alpha]1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-[alpha]1,3-Lac. The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of [alpha]-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the [alpha]-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides.