A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris

Abstract

To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N -terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N -glycosylation.

A Combined System for Engineering Glycosylation Efficiency and Glycan Structure in Saccharomyces cerevisiae

“…A simplification of the system was achieved by using a P. pastoris strain devoid of the ALG3 gene in the lipid-linked oligosaccharide (LLO) biosynthesis pathway, leading to the generation of a truncated N-glycan structure and necessitating the insertion of a ManI activity only (17). At the moment, terminally galactosylated or sialylated N-glycans are being produced in P. pastoris (18,19).…”

Glycoengineering of yeasts from the perspective of glycosylation efficiency

“…The architecture of Golgi-localized glycosyltransferases and mannosidases is highly conserved between species and consists of an N-terminal transmembrane domain (TMD) with an extension into the cytoplasm of different length, a stem domain and a C-terminal catalytic domain. In order to target vertebrate glycosyltransferases and mannosidases in the Golgi apparatus of yeast, fusions of TMD and stem domains of yeast mannosyltransferases with catalytic domains of vertebrate glycosyltransferases and mannosidases have been created, selecting for each enzyme activity the optimal TMD and stem domain [10].…”

“…Either ManI activity is destined to the early Golgi apparatus [10] or a fungal α1,2 mannosidase (α1,2-Man) is targeted into the ER and the Man3GlcNAc2 structure is already generated there [14] ( Figure 1C and D).…”

Glycoengineered Yeast as an Alternative Monoclonal Antibody Discovery and Production Platform

“…Besides OCH1 gene, other mannosylphosphate transferases and -mannosyltransferases can further modify the glycans at secreted proteins into fungal type of glycosylation, and to eliminate these genes is an essential step of engineering the yeast glycosylation pathways into human like. After eliminating yeast enzymes specific for fungal glycosylation modification, an efficient high throughput approach was developed by screening combinatorial libraries of fusing catalytic domain of modification enzymes with localization leader sequence (Choi et al, 2003;Nett et al, 2011). With this approach, -1,2 mannosidase (MNS1), N-acetylglucosaminyltransferase I (GNT1), mannosidase II (MNS2) and Nacetylglucosaminyl transferase II (GNT2) were engineered into Pichia pastoris and localized from early to later golgi in a sequential order.…”

“…Due to the high degree of selectivity of these agents, in particular for cancer targets, they can be designed to selectively target tumor cells and elicit a variety of responses. These agents can kill cells directly by carrying a toxic payload to the target or can orchestrate the destruction of cells by activating immune system components, blocking receptors or sequestering growth factors (Nicolaides et al, 2010).…”