Modification of the endoplasmic reticulum morphology enables improved recombinant antibody expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a versatile cell factory used for manufacturing of a wide range of products, among them recombinant proteins. Protein folding is one of the rate-limiting processes and this shortcoming is often overcome by the expression of folding catalysts and chaperones in the endoplasmic reticulum (ER). In this work, we aimed to establish the impact of ER structure on cellular productivity. The reticulon proteins Rtn1p and Rtn2p, and Yop1p are membrane curvature inducing proteins that define the morphology of the ER and depletion of these proteins creates yeast cells with a higher ER sheet-to-tubule ratio. We created yeast strains with different combinations of deletions of Rtn1p, Rtn2p, and Yop1p coding genes in cells with a normal or expanded ER lumen. We identified strains that reached up to 2.2-fold higher antibody titres compared to the control strain. The expanded ER membrane reached by deletion of the lipid biosynthesis repressor OPI1 was essential for the increased productivity. The improved specific productivity was accompanied by an up to 2-fold enlarged ER surface area and a 1.5-fold increased cross-sectional cell area. Furthermore, the strains with enlarged ER displayed an attenuated unfolded protein response. These results underline the impact that ER structures have on productivity and support the notion that reprogramming subcellular structures belongs into the toolbox of synthetic biology.

The Impact of Glycoengineering on the Endoplasmic Reticulum Quality Control System in Yeasts.

Yeasts are widely used and established production hosts for biopharmaceuticals. Despite of tremendous advances on creating human-type N-glycosylation, N-glycosylated biopharmaceuticals manufactured with yeasts are missing on the market. The N-linked glycans fulfill several purposes. They are essential for the properties of the final protein product for example modulating half-lives or interactions with cellular components. Still, while the protein is being formed in the endoplasmic reticulum, specific glycan intermediates play crucial roles in the folding of or disposal of proteins which failed to fold. Despite of this intricate interplay between glycan intermediates and the cellular machinery, many of the glycoengineering approaches are based on modifications of the N-glycan processing steps in the endoplasmic reticulum (ER). These N-glycans deviate from the canonical structures required for interactions with the lectins of the ER quality control system. In this review we provide a concise overview on the N-glycan biosynthesis, glycan-dependent protein folding and quality control systems and the wide array glycoengineering approaches. Furthermore, we discuss how the current glycoengineering approaches partially or fully by-pass glycan-dependent protein folding mechanisms or create structures that mimic the glycan epitope required for ER associated protein degradation.

Similarity Index for the Fat Fraction between Breast Milk and Infant Formulas.

The similarity of the fat fraction in infant formulas rich in either bovine milk fat (MF) or vegetable oil (VO) to breast milk was evaluated by analyzing their lipid composition. Milk fat-rich formulas were highly similar (average similarity index 0.68) to breast milk compared to the VO-rich formulas (average similarity index 0.56). The highest difference in the indices was found in the contents of cholesterol (0.66 vs 0.28 in MF- and VO-rich formulas, respectively, on average) and polar lipids (0.84 vs 0.53), the positional distribution of fatty acids in the sn-2 position of triacylglycerols (0.53 vs 0.28), and fatty acid composition (0.72 vs 0.54). The VO-based formulas were superior in similarity in n - 6 PUFA. Thus, the addition of bovine MF fractions is an effective way to increase the similarity between the lipid composition of infant formulas and human milk.

Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae.

N-glycosylation is an important posttranslational modification affecting the properties and quality of therapeutic proteins. Glycoengineering in yeast aims to produce proteins carrying human-compatible glycosylation, enabling the production of therapeutic proteins in yeasts. In this work, we demonstrate further development and characterization of a glycoengineering strategy in a Saccharomyces cerevisiae Δalg3 Δalg11 strain where a truncated Man3GlcNAc2 glycan precursor is formed due to a disrupted lipid-linked oligosaccharide synthesis pathway. We produced galactosylated complex-type and hybrid-like N-glycans by expressing a human galactosyltransferase fusion protein both with and without a UDP-glucose 4-epimerase domain from Schizosaccharomyces pombe. Our results showed that the presence of the UDP-glucose 4-epimerase domain was beneficial for the production of digalactosylated complex-type glycans also when extracellular galactose was supplied, suggesting that the positive impact of the UDP-glucose 4-epimerase domain on the galactosylation process can be linked to other processes than its catalytic activity. Moreover, optimization of the expression of human GlcNAc transferases I and II and supplementation of glucosamine in the growth medium increased the formation of galactosylated complex-type glycans. Additionally, we provide further characterization of the interfering mannosylation taking place in the glycoengineered yeast strain. KEY POINTS: • Glycoengineered Saccharomyces cerevisiae can form galactosylated N-glycans. • Genetic constructs impact the activities of the expressed glycosyltransferases. • Growth medium supplementation increases formation of target N-glycan structure.

Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis.

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.

Mining Data From Plasma Cell Differentiation Identified Novel Genes for Engineering of a Yeast Antibody Factory.

Saccharomyces cerevisiae is a common platform for production of therapeutic proteins, but it is not intrinsically suited for the manufacturing of antibodies. Antibodies are naturally produced by plasma cells (PCs) and studies conducted on PC differentiation provide a comprehensive blueprint for the cellular transformations needed to create an antibody factory. In this study we mined transcriptomics data from PC differentiation to improve antibody secretion by S. cerevisiae. Through data exploration, we identified several new target genes. We tested the effects of 14 genetic modifications belonging to different cellular processes on protein production. Four of the tested genes resulted in improved antibody expression. The ER stress sensor IRE1 increased the final titer by 1.8-fold and smaller effects were observed with PSA1, GOT1, and HUT1 increasing antibody titers by 1. 6-, 1. 4-, and 1.4-fold. When testing combinations of these genes, the highest increases were observed when co-expressing IRE1 with PSA1, or IRE1 with PSA1 and HUT1, resulting in 3.8- and 3.1-fold higher antibody titers. In contrast, strains expressing IRE1 alone or in combination with the other genes produced similar or lower levels of recombinantly expressed endogenous yeast acid phosphatase compared to the controls. Using a genetic UPR responsive GFP reporter construct, we show that IRE1 acts through constitutive activation of the unfolded protein response. Moreover, the positive effect of IRE1 expression was transferable to other antibody molecules. We demonstrate how data exploration from an evolutionary distant, but highly specialized cell type can pinpoint new genetic targets and provide a novel concept for rationalized cell engineering.

Investigating the role of ERAD on antibody processing in glycoengineered Saccharomyces cerevisiae.

N-glycosylation plays an important role in the endoplasmic reticulum quality control (ERQC). N-glycan biosynthesis pathways have been engineered in yeasts and fungi to enable the production of therapeutic glycoproteins with human-compatible N-glycosylation, and some glycoengineering approaches alter the synthesis of the lipid-linked oligosaccharide (LLO). Because the effects of LLO engineering on ERQC are currently unknown, we characterized intracellular processing of IgG in glycoengineered Δalg3 Δalg11 Saccharomyces cerevisiae strain and analyzed how altered LLO structures affect endoplasmic reticulum-associated degradation (ERAD). Intracellular IgG light and heavy chain molecules expressed in Δalg3 Δalg11 strain are ERAD substrates and targeted to ERAD independently of Yos9p and Htm1p, whereas in the presence of ALG3 ERAD targeting is dependent on Yos9p but does not require Htm1p. Blocking of ERAD accumulated ER and post-Golgi forms of IgG and increased glycosylation of matα secretion signal but did not improve IgG secretion. Our results show ERAD targeting of a heterologous glycoprotein in yeast, and suggest that proteins in the ER can be targeted to ERAD via other mechanisms than the Htm1p-Yos9p-dependent route when the LLO biosynthesis is altered.

Production and characterization of Aspergillus niger GH29 family α-fucosidase and production of a novel non-reducing 1-fucosyllactose.

Fucosylated oligosaccharides are interesting molecules due to their bioactive properties. In particular, their application as active ingredient in milk powders is attractive for dairy industries. The objective of this study was to characterize the glycosyl hydrolase family 29 α-fucosidase produced by Aspergillus niger and test its ability to transfucosylate lactose with a view towards potential industrial applications such as the valorization of the lactose side stream produced by dairy industry. In order to reduce costs and toxicity the use of free fucose instead of environmentally questionable fucose derivatives was studied. In contrast to earlier studies, a recombinantly produced A. niger α-fucosidase was utilized. Using pNP-fucose as substrate, the optimal pH for hydrolytic activity was determined to be 3.8. The optimal temperature for a 30-min reaction was 60 °C, and considering temperature stability, the optimal temperature for a 24-h reaction was defined as 45 °C For the same hydrolysis reaction, the kinetic values were calculated to be 0.385 mM for the KM and 2.8 mmol/(mg*h) for the Vmax. Transfucosylation of lactose occurred at high substrate concentrations when reaction time was elongated to several days. The structure of the product trisaccharide was defined as 1-fucosyllactose, where fucose is α-linked to the anomeric carbon of the ß-glucose moiety of lactose. Furthermore, the enzyme was able to hydrolyze its own transfucosylation product and 2'-fucosyllactose but only poorly 3-fucosyllactose. As a conclusion, α-fucosidase from A. niger can transfucosylate lactose using free fucose as substrate producing a novel non-reducing 1-fucosyllactose.

Tailoring N-Glycan Biosynthesis for Production of Therapeutic Proteins in Saccharomyces cerevisiae.

The ability to control and adjust the N-glycosylation pathway of Saccharomyces cerevisiae is a key step toward production of therapeutic glycoproteins such as antibodies or erythropoietin. The focus of this chapter is to describe the road from yeast-type N-glycosylation to human-type complex N-glycosylation. The chapter describes the cell engineering and provides the detailed analytical procedures required to perform glycan analysis using MALDI-TOF mass spectrometry.

Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach.

The cellular changes induced by heterologous protein expression in the yeast Saccharomyces cerevisiae have been analysed on many levels and found to be significant. However, even though high-level protein production poses a metabolic burden, evaluation of the expression host at the level of the metabolome has often been neglected. We present a comparison of metabolite profiles of a wild-type strain with those of three strains producing recombinant antibody variants of increasing size and complexity: an scFv fragment, an scFv-Fc fusion protein and a full-length IgG molecule. Under producing conditions, all three recombinant strains showed a clear decrease in growth rate compared with the wild-type strain and the severity of the growth phenotype increased with size of the protein. The levels of 76 intracellular metabolites were determined using a targeted (semi) quantitative mass spectrometry based approach. Based on unsupervised and supervised multivariate analysis of metabolite profiles, together with pathway activity profiling, the recombinant strains were found to be significantly different from each other and from the wild-type strain. We observed the most prominent changes in metabolite levels for metabolites involved in amino acid and redox metabolism. Induction of the unfolded protein response was detected in all producing strains and is considered to be a contributing factor to the overall metabolic burden on the cells.

Enzymatic synthesis of fucose-containing galacto-oligosaccharides using ß-galactosidase and identification of novel disaccharide structures.

Fucosylated oligosaccharides have an important role in maintaining a healthy immune system and homeostatic gut microflora. This study employed a commercial ß-galactosidase in the production of fucose-containing galacto-oligosaccharides (fGOS) from lactose and fucose. The production was optimized using experiment design and optimal conditions for a batch production in 3-liter scale. The reaction product was analyzed and the produced galactose-fucose disaccharides were purified. The structures of these disaccharides were determined using NMR and it was verified that one major product with the structure Galß1-3Fuc and two minor products with the structures Galß1-4Fuc and Galß1-2Fuc were formed. Additionally, the product composition was defined in more detail using several different analytical methods. It was concluded that the final product contained 42% total monosaccharides, 40% disaccharides and 18% of larger oligosaccharides. 290 µmol of fGOS was produced per gram of reaction mixture and 37% of the added fucose was bound to fGOS. The fraction of fGOS from total oligosaccharides was determined as 44%. This fGOS product could be used as a new putative route to deliver fucose to the intestine.

Following nature's roadmap: folding factors from plasma cells led to improvements in antibody secretion in S. cerevisiae.

Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full-length antibodies. Despite numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast. A subset of mammalian folding factors upregulated in plasma cells for expression in yeast and screened for beneficial effects on antibody secretion using a high-throughput ELISA platform was selected. Co-expression of the mammalian chaperone BiP, the co-chaperone GRP170, or the peptidyl-prolyl isomerase FKBP2, with the antibody improved specific product yields up to two-fold. By comparing strains expressing FKBP2 or the yeast PPIase Cpr5p, the authors demonstrate that speeding up peptidyl-prolyl isomerization by upregulation of catalyzing enzymes is a key factor to improve antibody titers in yeast. The findings show that following the route of plasma cells can improve product titers and contribute to developing an alternative yeast-based antibody factory.

Screening for novel genes of Saccharomyces cerevisiae involved in recombinant antibody production.

Cost-effective manufacturing of biopharmaceuticals in non-mammalian hosts still requires tremendous efforts in strain development. In order to expedite identification of novel leads for strain engineering, we used a transposon-mutagenized yeast genomic DNA library to create a collection of Saccharomyces cerevisiae deletion strains expressing a full-length IgG antibody. Using a high-throughput screening, transformants with either significantly higher or lower IgG expression were selected. The integration site of the transposon in three of the selected strains was located by DNA sequencing. The inserted DNA lay within the VPS30 and TAR1 open reading frame, and upstream of the HEM13 open reading frame. The complete coding sequence of these genes was deleted in the wild-type strain background to confirm the IgG expression phenotypes. Production of recombinant antibody was increased 2-fold in the Δvps30 strain, but only mildly affected secretion levels in the Δtar1 strain. Remarkably, expression of endogenous yeast acid phosphatase was increased 1.7- and 2.4-fold in Δvps30 and Δtar1 strains. The study confirmed the power of genome-wide high-throughput screens for strain development and highlights the importance of using the target molecule during the screening process.

Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum.

BACKGROUND: The yeast Saccharomyces cerevisiae provides intriguing possibilities for synthetic biology and bioprocess applications, but its use is still constrained by cellular characteristics that limit the product yields. Considering the production of advanced biopharmaceuticals, a major hindrance lies in the yeast endoplasmic reticulum (ER), as it is not equipped for efficient and large scale folding of complex proteins, such as human antibodies. RESULTS: Following the example of professional secretory cells, we show that inducing an ER expansion in yeast by deleting the lipid-regulator gene OPI1 can improve the secretion capacity of full-length antibodies up to fourfold. Based on wild-type and ER-enlarged yeast strains, we conducted a screening of a folding factor overexpression library to identify proteins and their expression levels that enhance the secretion of antibodies. Out of six genes tested, addition of the peptidyl-prolyl isomerase CPR5 provided the most beneficial effect on specific product yield while PDI1, ERO1, KAR2, LHS1 and SIL1 had a mild or even negative effect to antibody secretion efficiency. Combining genes for ER enhancement did not induce any significant additional effect compared to addition of just one element. By combining the Δopi1 strain, with the enlarged ER, with CPR5 overexpression, we were able to boost the specific antibody product yield by a factor of 10 relative to the non-engineered strain. CONCLUSIONS: Engineering protein folding in vivo is a major task for biopharmaceuticals production in yeast and needs to be optimized at several levels. By rational strain design and high-throughput screening applications we were able to increase the specific secreted antibody yields of S. cerevisiae up to 10-fold, providing a promising strain for further process optimization and platform development for antibody production.

A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae.

N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.

Analysis of antibody production in Saccharomyces cerevisiae: effects of ER protein quality control disruption.

One of the main limitations for heterologous protein production in the yeast Saccharomyces cerevisiae is the protein-folding capacity in the endoplasmic reticulum (ER). Accumulation of unfolded proteins triggers the unfolded protein response (UPR), which resolves the stress by increasing the capacity for protein folding and removal of unfolded proteins by the ER-associated degradation (ERAD) system. In order to analyze the influence of ERAD on production of a human IgG, we disrupted ERAD at different stages by deletion of the HTM1, YOS9, HRD1, HRD3, or UBC7 gene, with or without a disruption of the UPR by deletion of the IRE1 gene. All deletion strains were viable and did not exhibit a growth phenotype under normal growth conditions. Deletion of HTM1 resulted in a small increase in antibody production, whereas a small decrease in antibody production was observed in the Δhrd1, Δhrd3, and Δubc7 yeast strains, and a stronger decrease in the Δyos9 yeast strain. Deletion of the IRE1 gene had contrasting effects in the ERAD mutants, with a strongly decreased production in wild-type cells and partially reversed effects in combination with the Δhtm1 or the Δyos9 deletions. In order to study IgG clearance from the ER, an assay was developed using the inhibitory effect of glucose on the GAL1 promoter that is driving IgG expression. The Δyos9Δire1and Δhtm1Δire1 strains showed a delayed IgG clearance from the cells, showing that removal of components for the generation and recognition of the glycan signal needed for ERAD-mediated protein degradation might increase the IgG ER residence time.

Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly.

Protein degradation is essential for cellular homeostasis. We developed a sensitive approach to examining protein degradation rates in Saccharomyces cerevisiae by coupling a SILAC approach to selected reaction monitoring (SRM) mass spectrometry. Combined with genetic tools, this analysis made it possible to study the assembly of the oligosaccharyl transferase complex. The ER-associated degradation machinery compensated for disturbed homeostasis of complex components by degradation of subunits in excess. On a larger scale, protein degradation in the ER was found to be a minor factor in the regulation of protein homeostasis in exponentially growing cells, but ERAD became relevant when the gene dosage was affected, as demonstrated in heterozygous diploid cells. Hence the alleviation of fitness defects due to abnormal gene copy numbers might be an important function of protein degradation.

Homologous recombinatorial cloning without the creation of single-stranded ends: exonuclease and ligation-independent cloning (ELIC).

We describe a new type of molecular cloning that complements the available strategies for homologous recombinatorial cloning. Purified, linear double-stranded DNA molecules with homologous ends are simply mixed in water and they transform readily into E. coli. Insert and linear vector need as few as ten base pairs of homologous sequence at their ends and essentially no incubation or enzyme treatments are needed for creating recombinants from linear fragments. Our method outcompetes most existing cloning methods in simplicity and affordability and is well-suited for high-throughput applications.

An enzyme-based screening system for the rapid assessment of protein N-glycosylation efficiency in yeast.

N-Glycosylation efficiency is a key parameter when studying components of the protein N-glycosylation pathway, but was recently also recognized as an important factor in the production of glycosylated proteins. We have developed a novel assay to quantify N-glycosylation efficiency of proteins. This assay is based on the secreted activity of yeast acid phosphatase, the proper folding and hence secretion of which is strongly dependent on its N-glycosylation status. The results show that the reporter yields a quantitative measure for protein N-glycosylation in yeast, which is in good agreement with classically used assay based on protein migration patterns on SDS-PAGE. However, the assay is less laborious and is adaptable to high-throughput screening approaches as exemplified.

Glycoengineering of yeasts from the perspective of glycosylation efficiency.

N-linked glycosylation of proteins is one of the most common posttranslational modifications. N-glycan structures and N-glycosylation efficiency are crucial parameters in the production of N-glycosylated proteins. Yeast cells can be seen as an attractive production host for therapeutic glycoproteins and pioneering work of glycoengineering was performed in Pichia pastoris, realizing yeast strains capable of producing defined, human-type N-glycans. Most strategies used for glycoengineering rely on the modification of the lipid-linked oligosaccharide biosynthesis for the generation of the substrate for Golgi-localized glycosyltransferases. However, modifications in the lipid-linked oligosaccharide biosynthesis often result in the accumulation of intermediate structures and cause hypoglycosylation of client proteins. In order to ensure complete N-glycosylation, the flow of lipid-linked oligosaccharide through the biosynthetic pathway and the transfer of the oligosaccharide from the donor lipid onto the protein have to be optimized. A promising tool to improve site occupancy is the expression of protozoan oligosaccharyltransferases, which possess altered specificities for the oligosaccharide and also for the protein acceptor site. Furthermore, flipping of the lipid-linked oligosaccharide into the ER lumen can be improved by overexpression of an artificial flippase. Improving the glycosylation efficiency ensures that not only homogeneous N-glycan structures are generated, but also client proteins are fully glycosylated.