Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope

Anne M Ruffing,Rachel R Chen

doi:10.1186/1475-2859-9-1

Abstract

BackgroundOligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-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 α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac.ResultsAgrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-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-α1,3-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-α1,3-Lac.ConclusionsThe Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-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.

Highlights

Oligosaccharides containing a terminal Gal-a1,3-Gal moiety are collectively known as a-Gal epitopes. a-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments
We have previously demonstrated that Agrobacterium sp. strain ATCC 31749 is a good host for oligosaccharide production through the synthesis of b1,4-Gal disaccharides [15]
In order to utilize uridine diphosphate (UDP)-glucose for synthesis of Gal-a1,3-Lac, two additional enzymes are required: a UDP-galactose 4’-epimerase and an a1,3-galactosyltransferase (Figure 1)

Summary

Introduction

Oligosaccharides containing a terminal Gal-a1,3-Gal moiety are collectively known as a-Gal epitopes. a-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The a1,3-GalT was inactivated in ancestral Old World primates approximately 20-28 million years ago, resulting in the absence of a-Gal epitopes in humans, apes, and Old World monkeys today [1,2] These evolutionary descendents of Old World primates produce an antibody to Gal-a1,3-Gal-containing oligosaccharides known as anti-Gal. Anti-Gal is the most abundant natural antibody in humans, and as a result, exposure to aGal epitopes generates a strong immune response [3]. Uptake can be greatly enhanced by the presence of an IgG antibody, such as anti-Gal, bound to its associated antigen Based on this principle, several vaccines have been modified with a-Gal epitopes in an effort to improve vaccine uptake and efficacy. The promising results of these a-Gal-based treatments have stimulated the demand for a-Gal epitope production

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