The Research Of Glycosylation Engineering In Kluyveromyces Lactis

Glycoprotein, including antibody, is becoming the majority of biotechnological drug. But its production ability is too low to fulfill its fast increasing demands. To date, mammalian cell culture is the only process to yield human-like glycoproteins. However, its high production costs, requirement for complex media and concern of viral contamination have inhibited the development of glycoprotein drug industry. Yeast as one of the simple eukaryotes has been widely used in the production of recombinant proteins, owing to its ability to grow on chemically defined media in the absence of animal-derived proteins, ease of scale-up, and high yields of secreted protein. But glycoproteins derived from yeast always contain hyper-mannosylated glycans, which are immunogenic in human, so it limits their therapeutic value. To solve this problem, the glycoprotein engineering of yeast is to reconstruct a mammalian cell-like glycan synthesis passway in yeast. Most of the yeast glycosylation engineering reseasches were in Saccharomyces cerevisiae and Pichia pastoris. Little has been concerned in Kluyveromyces lactis, which is one of the four majoritic yeast expression hosts. Kluyveromyces lactis is an attractive expression host, for its safety, easily genetical manipulation, fully sequenced genome, and high titer of heterologous proteins. So, glycosylation engineering in yeast Kluyveromyces lactis was carried out firstly in this study.1. An auxotrophic host strain was constructed for molecular genetic manipulation based on KlURA3 selectable marker in Kluyveromyces lactis. The K. lactis strain ATCC8585, a prototrophic natural isolate, was used as the host strain and a two-step homologous recombination method was developed. A Klura3Δmutant strain was constructed. Using this stain and the method, a new technique to recyclely use of the ura3 marker was developed, which satisfy with the demands of many selectable markers required in the glycoengineering.2. The expression system characterized with non-hyperglycosylation was generated based on K. lactis och1Δand mnn1Δdouble mutant strain. The KlOCH1 gene, which encoding theα-1,6-mannosyltransferase, and the KlMNN1 gene, which putative encoding theα-1,3-mannosyltransferase were knockouted The glycoproteins of HSA/GM-CSF derived from the K. lactis wild-type, Kloch1Δand Kloch1 mnn1Δdouble strains were purified and analyzed by SDS-PAGE. HSA/GM-CSF with yeast-type hyperglycosylation was observed by coomassie brilliant blue staining in K. lactis wild-type strain, while the homogeneous lower-glycosylation in mutant stains.3. The N-glycan of ER-core Man9GlcNAc2 of glycoprotein HSA/GM-CSF was firstly obtained in recombinant yeast K. lactis. In order to estimate the function of KlOCH1 and KlMNN1 on N-glycosylation, the structures of N-glycan assembled on HSA/GM-CSF secreted from the K. lactis wild-type, Kloch1Δand Kloch1 mnn1Δdouble mutant strains were analyzed by FACE (fluorophore-assisted carbohydrate electrophoresis) and DSA-FACE. Man > 30GlcNAc2 oligosaccharide of secreted glycoprotein HSA/ GM-CSF was observed in wild-type, while Man13~14GlcNAc2 in Kloch1Δmutant and Man9~11GlcNAc2 in Kloch1 mnn1Δdouble mutant strain. The results showed that the KlOCH1 plays a key role in the addition mannose to the core oligosaccharide. The hypotheticalα-1,3-mannosyltransferase KlMNN1 is also proved to contribute to the generation of hyper-mannosylation in yeast K. lactis.4. The mammalian cells-like mannose-type (Man5GlcNAc2) sugar chain was produced in yeast K. lactis firstly A library contained catalytic domains ofα-1,2-mannosidases (MANI) from Arabidopsis thaliana, Trichoderma reesei, Homo sapiens, and contained the leaders encoding N-terminal peptides of known type II membrane proteins that either localize in the ER of S. cerevisiae and K. lactis was constructed. A double mutant K. lactis lacking two mannosyltransferase activities was then transformed with anα-1,2-mannosidase expression vector. In the K. lactis expressed chimera of ER-ScMNSI-human MANI(Δ63), the N-glycan of Man5GlcNAc2, and Man9～11GlcNAc2 in secreted glycoprotein HSA/GM-CSF were observed, while in the K. lactis expressed chimera of ScSEC12- ATMANI(Δ48) N-glycans of Man5GlcNAc2, and Man6～11GlcNAc2 were produced. In the case of the strain expressed ER-ScMNSI-ATMANI(Δ48) chimera, a major band corresponding to Man6GlcNAc2 and minor bands Man7~8GlcNAc2 were obstained.In summary, glycosylation engineering was carried out firstly in yeast K. lactis. The Kloch1 mnn1Δdouble strain was constructed to express the glycoprotein with homogeneous lower-glycosylation after the KlOCH1 and KlMNN1 were knockouted successfully. The mammalian cells-like mannose-type (Man5GlcNAc2) sugar chain was produced in recombinant yeast K. lactis. The ura3 selectable marker for easily genetically manipulating, and the method DSA-FACE for the analysis of N-glycans could be utilized in glycosylation engineering.This is the first report that a recombinant K. lactis was able to produce Man5GlcNAc2 core oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains. It is important to generate Man5GlcNAc2 structures in the secretory pathway in vivo for glycoengineering. The generation of Man5GlcNAc2 structures presents it realizable in yeast K. lactis, and supply for the substrate to be transformed to hybrid- and complex-type glcans.