Genetic and process engineering strategies for enhanced recombinant N-glycoprotein production in bacteria

Microbial Cell Factories

Dennis Linton¹  Neil Dixon²  Fenryco Pratama³ 

[1]Faculty of Biology, Medicine and Health, The University of Manchester, M1 7DN, Manchester, UK

[2]Manchester Institute of Biotechnology (MIB), The University of Manchester, M1 7DN, Manchester, UK

[3]Department of Chemistry, The University of Manchester, M1 7DN, Manchester, UK

[4]Manchester Institute of Biotechnology (MIB), The University of Manchester, M1 7DN, Manchester, UK

[5]Department of Chemistry, The University of Manchester, M1 7DN, Manchester, UK

[6]Microbial Biotechnology Research Group, School of Life Sciences and Technology, Institut Teknologi Bandung, 40132, Bandung, Indonesia

关键词: Glycoengineering;    N;    Bacterial host engineering;    Protein folding;    Process optimisation;   

DOI  :  10.1186/s12934-021-01689-x

来源: Springer

【 摘 要 】

BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation, and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements, is the impact of target protein sequon accessibility during glycosylation.ResultsHere, we explore a series of genetic and process engineering strategies to increase recombinant N-linked glycosylation, mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductase or disulphide-bond isomerase activity. These approaches achieve up to twofold improvement in glycosylation efficiency. Furthermore, we also demonstrate that supplementation with the chemical oxidant cystine enhances the titre of glycoprotein in an oxidoreductase knockout strain by improving total protein production and cell fitness, while at the same time maintaining higher levels of glycosylation efficiency.ConclusionsIn this study, we demonstrate that improved protein glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native host such as Campylobacter jejuni and mammalian cells. Furthermore, it provides insight into strain engineering and bioprocess strategies, to improve glycoprotein yield and titre, and to avoid physiological burden of unfolded protein stress upon cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production.

【 授权许可】

CC BY