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

Ruffing, Chen

Description

Oligosaccharides 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. Results Agrobacterium 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. Conclusions The 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.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2010
• Nombre de lectures: 1
• Langue: English

Extrait

Ruffing and ChenMicrobial Cell Factories2010,9:1 http://www.microbialcellfactories.com/content/9/1/1

R E S E A R C HOpen Access Metabolic engineering ofAgrobacteriumsp. strain ATCC 31749 for production of anaGal epitope * Anne M Ruffing, Rachel R Chen

Abstract Background:Oligosaccharides containing a terminal Gala1,3Gal moiety are collectively known asa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 difficulty associated with synthesizing theaGal epitope hinders the development and application of these treatments due to the limited availability and high cost of theaGal epitope. This work illustrates the development of a wholecell biocatalyst for synthesizing theaGal epitope, Gal a1,3Lac. Results:Agrobacteriumsp. ATCC 31749 was engineered to produce Gala1,3Lac by the introduction of a UDP galactose 4’epimerase:a1,3galactosyltransferase fusion enzyme. The engineeredAgrobacteriumsynthesized 0.4 g/L of theaGal epitope. Additional metabolic engineering efforts addressed the factors limitinga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a1,3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a1,3Lac. Conclusions:TheAgrobacteriumbiocatalyst developed in this work synthesizes gramscale quantities ofaGal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of theaGal epitope. Furthermore, the engineeredAgrobacterium, with increased lactose uptake and improved UDPglucose availability, is a promising host for the production of other medicallyrelevant oligosaccharides.

Background aGal epitopes are oligosaccharides containing terminal Gala1,3Gal residues. In nature, three mainaGal epi topes are produced: two trisaccharides (Gala1,3Gal b1,4GlcNAc and Gala1,3Lac) and a pentasaccharide (Gala1,3Galb1,4GlcNAcb1,3Galb1,4Glc). These epitopes are components of glycolipids and glycopro teins displayed on the cell surface of nonprimate mam mals and New World monkeys via expression of an a1,3galactosyltransferase (a1,3GalT). Thea1,3GalT was inactivated in ancestral Old World primates approximately 2028 million years ago, resulting in the absence ofaGal epitopes in humans, apes, and Old World monkeys today [1,2]. These evolutionary

* Correspondence: [e-mail] School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 303320100, USA

descendents of Old World primates produce an anti body to Gala1,3Galcontaining oligosaccharides known as antiGal. AntiGal is the most abundant nat ural antibody in humans, and as a result, exposure toa Gal epitopes generates a strong immune response [3]. Many current research efforts exploit the human immune response toaGal epitopes. The efficacy of a vaccine is often determined by uptake of the vaccine by antigen presenting cells. 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 withaGal epitopes in an effort to improve vaccine uptake and efficacy. This strategy was applied to flu and HIV vaccines and was found to be more effective than the nonmodified vac cine in animal studies [4,5]. In addition to enhancing vaccine efficacy, the immunogenicity ofaGal epitopes

© 2010 Ruffing and Chen; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.