Engineering a C-mannosylation pathway into Pichia pastoris

A John1, N Scott2 and ED Goddard-Borger1

  1. Chemical Biology Division, The Walter and Eliza Hall Institute, Parkville, Victoria, Australia
  2. Department of Microbiology and Immunology, Doherty Institute, University of Melbourne, Parkville, Victoria, Australia

Producing recombinant proteins on an industrial scale with human-like patterns of glycosylation remains as an important challenge in biotechnology. Heterologous expression of proteins in mammalian cells is expensive relative to other expression systems and is complicated by its susceptibility to viral contamination. Yeast protein production systems are key to delivering low-cost biologics because they are inexpensive to culture but maintain the benefits of a eukaryotic expression host. However, yeast glycosylation pathways are significantly different to those in humans, which limits its usefulness for manufacturing biologics. This problem has driven significant efforts to 'humanise' glycosylation pathways in yeast. While efforts have mainly focused on producing proteins with homogenous human-like N-glycans, engineering other mammalian glycosylation pathways into yeast remains an important goal. Tryptophan C-mannosylation is an unusual co-translational modification found in metazoans. This modification occurs on the WXXW consensus motif in thrombospondin repeats (TSRs) and type-I cytokine receptors, as well as some cytokines, hyaluronidases, RNAses, and virus glycoproteins. Establishing yeast strains capable of Trp-C-mannosylation would facilitate the low cost production of proteins that could be useful as biologics or vaccines. Yeast possesses no detectable C-mannosyltransferase activity, but they do produce a large quantity of mannose phosphate dolichol, the substrate utilized by C-mannosyltransferases. We engineered Pichia pastoris to constitutively express the C. elegans C-mannosyltransferase localised to the ER. This heterologous enzyme harnessed intracellular mannose phosphate dolichol to drive C-mannosylation of proteins over-expressed in this system. This refinement in the 'humanisation' of yeast glycosylation pathways also provides a means to characterise the role and properties of this poorly understood protein modification.