Wheat pathogen -myristoyltransferase inhibitors: on-target antifungal activity and an unusual metabolic defense mechanism.

is the causative agent of blotch (STB), which costs billions of dollars annually to major wheat-producing countries in terms of both fungicide use and crop loss. Agricultural pathogenic fungi have acquired resistance to most commercially available fungicide classes, and the rate of discovery and development of new fungicides has stalled, demanding new approaches and insights. Here we investigate a potential mechanism of targeting an important wheat pathogen inhibition of -myristoyltransferase (NMT).

Engineering Pathways in Central Carbon Metabolism Help to Increase Glycan Production and Improve -Type Glycosylation of Recombinant Proteins in .

strains have been modified in a variety of ways to enhance the production of different recombinant proteins, targeting membrane protein expression, proteins with disulphide bonds, and more recently, proteins which require -linked glycosylation. The addition of glycans to proteins remains a relatively inefficient process and here we aimed to combine genetic modifications within central carbon metabolic pathways in order to increase glycan precursor pools, prior to transfer onto polypeptide backbones. Using a lectin screen that detects cell surface representation of glycans, together with Western blot analyses using an -antigen ligase mutant strain, the enhanced uptake and phosphorylation of sugars () from the media combined with conservation of carbon through the glyoxylate shunt () improved glycosylation efficiency of a bacterial protein AcrA by 69% and over 100% in an engineered human protein IFN-α2b.

Producing a glycosylating Escherichia coli cell factory: The placement of the bacterial oligosaccharyl transferase pglB onto the genome.

Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform.

Generation of Recombinant N-Linked Glycoproteins in E. coli.

The production of N-linked recombinant glycoproteins is possible in a variety of biotechnology host cells, and more recently in the bacterial workhorse, Escherichia coli. This methods chapter will outline the components and procedures needed to produce N-linked glycoproteins in E. coli, utilizing Campylobacter jejuni glycosylation machinery, although other related genes can be used with minimal tweaks to this methodology.

Inverse Metabolic Engineering for Enhanced Glycoprotein Production in Escherichia coli.

Inverse metabolic engineering (IME) provides a strategy to rapidly identify the genetic elements responsible for the desired phenotype of a chosen target organism. This methodology has been successfully applied towards enhancing the N-linked glycosylation efficiency of Escherichia coli. Here, we describe the generation of differentially sized libraries from the E.

Escherichia coli as a glycoprotein production host: recent developments and challenges.

Chinese Hamster Ovary cells are the most popular host expression system for the large-scale production of human therapeutic glycoproteins, but, the race to engineer Escherichia coli to perform glycosylation is gathering pace. The successful functional transfer of an N-glycosylation pathway from Campylobacter jejuni to Escherichia coli in 2002 can be considered as the crucial first engineering step. Here, we discuss the recent advancements in the field of N-glycosylation of recombinant therapeutic proteins in E.