Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.

As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation.

The secretory pathway of Pichia pastoris was genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating α-1,6-mannosyltransferase gene from P. pastoris, several combinatorial genetic libraries were constructed to localize active α-1,2-mannosidase and human β-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of α-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modest in vivo [i.e., low levels of mannose (Man)5-(GlcNAc)2] activity, we were able to isolate several yeast strains that produce almost homogenous N-glycans of the (Man)5-(GlcNAc)2 type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)5-(GlcNAc)2 in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)5-(GlcNAc)2 as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation

The cluster of three genes, ACR1, ACR2, and ACR3, previously was shown to confer arsenical resistance in Saccharomyces cerevisiae. The overexpression of ACR3 induced high level arsenite resistance. The presence of ACR3 together with ACR2 on a multicopy plasmid was conducive to increased arsenate resistance. The function of ACR3 gene has now been investigated. Amino acid sequence analysis of Acr3p showed that this hypothetical protein has hydrophobic character with 10 putative transmembrane spans and is probably located in yeast plasma membrane. We constructed the acr3 null mutation. The resulting disruptants were 5-fold more sensitive to arsenate and arsenite than wild-type cells. The acr3 disruptants showed wild-type sensitivity to antimony, tellurite, cadmium, and phenylarsine oxide. The mechanism of arsenical resistance was assayed by transport experiments using radioactive arsenite. We did not observe any significant differences in the accumulation of 76 AsO 3 3؊ in wild-type cells, acr1 and acr3 disruptants. However, the high dosage of ACR3 gene resulted in loss of arsenite uptake. These results suggest that arsenite resistance in yeast is mediated by an arsenite transporter (Acr3p).Arsenicals are toxic compounds, which are commonly present in the environment at increasing concentrations as a result of industrial pollution (1). The pentavalent arsenate is a phosphate analog which interferes with phosphorylation reactions and competes with phosphate in transport (2-4). The more potent trivalent arsenite reacts with the sulfhydryl groups of proteins and inhibits many biochemical pathways (3, 5). Both arsenic salts were observed to induce morphological transformation and some cytogenetic effects (6 -8).Arsenical resistance phenomenon was described in many organisms from bacteria to mammalian cells (9 -13). Resistance to arsenate, arsenite, and antimonite in prokaryota is mediated by a plasmid-encoded transport system (14 -16). The Escherichia coli ars operon located on the plasmid R773 consists of five genes : arsR, arsD, arsA, arsB, and arsC (14, 17). The arsR and arsD encode trans-acting regulatory proteins (17,18). The products of arsA and arsB genes are the two subunits of an ATP-coupled oxyanion pump (14). The ArsA protein is the catalytic subunit exhibiting an ATPase activity (19). The ArsB protein is an inner membrane component of the pump (20), which acts as an anion channel and an anchor for the ArsA protein (21). The arsC gene was shown to encode an arsenate reductase (22). Similar ars operons were identified in Grampositive bacteria: Staphylococcus aureus (pI258) (15) and Staphylococcus xylosus (pSX267) (16). In staphylococcal ars operons arsR, -B, -C genes are conserved but arsD and arsA are absent. In this case arsenite efflux is mediated only by the ArsB protein coupled to the protonmotive force (23). Arsenical resistance in eukaryotic cells is also transport-mediated (4, 11, 13, 24). The existence of an energy-dependent arsenical efflux pump was demonstrated in Leishmania tarentolae (24, 25) an...