The humanization of N-glycosylation pathways in yeast

Yeast and other fungal protein-expression hosts have been extensively used to produce industrial enzymes, and are often the expression system of choice when manufacturing costs are of primary concern. However, for the production of therapeutic glycoproteins intended for use in humans, yeast have been less useful owing to their inability to modify proteins with human glycosylation structures. Yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and thereby compromises the efficacy of most therapeutic glycoproteins. Several approaches to humanizing yeast N-glycosylation pathways have been attempted over the past decade with limited success. Recently however, advances in the glycoengineering of yeast and the expression of therapeutic glycoproteins with humanized N-glycosylation structures have shown significant promise - this review summarizes the most important developments in the field.     

Production of therapeutic glycoproteins through the engineering of glycosylation pathway in yeast

The application of recombinant DNA technology to restructure metabolic networks can change metabolite and protein products by altering the biosynthetic pathways in an organism. Although some success has been achieved, a more detailed and thorough investigation of this approach is certainly warranted since it is clear that such methods hold great potential based on the encouraging results obtained so far. In last decade, there have been tremendous advances in the field of glycobiology and the stage has been set for the biotechnological production of glycoproteins for therapeutic use. Today glycoproteins are one of the most important groups of pharmaceutical products. In this study the attempt was made to focus on identifying technologies that may have general application for modifying glycosylation pathway of the yeast cells in order to produce glycoproteins of therapeutic use. The carbohydrates of therapeutic recombinant glycoproteins play very important roles in determining their pharmacokinetic properties. A number of biological interactions and biological functions mediated by glycans are also being targeted for therapeutic manipulationin vivo. For a commercially viable production of therapeutic glycoproteins a metabolic engineering of a host cell is yet to be established.     

Optogenetic switches for light-controlled gene expression in yeast

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

     

Engineering of protein secretion in yeast: strategies and impact on protein production

Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.     

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.     

A systems-level approach for metabolic engineering of yeast cell factories

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories.     

Expression of synthetic thaumatin genes in yeast

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.     

Engineering yeast for high level expression.

As a eukaryotic microbe, yeast remains an attractive host for the expression of a large variety of foreign proteins, including viral antigens, enzymes used as food additives and therapeutic agents. Important progress has been made in the understanding of the critical parameters influencing product yield, and a number of novel tools for the genetic engineering of powerful yeast expression systems have been developed. This review focuses on recent findings in foreign gene expression in the yeasts Saccharomyces, Pichia, Hansenula, and Kluyveromyces.     

Stability of recombinant plasmids in yeast

Yeasts represent one class of host for the production of recombinant proteins. Heterologous DNA is usually introduced into yeast strains in the form of multi-copy plasmids. During production, protein expression levels and rates are often limited by the stability of the recombinant organism. In this paper, we review the major factors affecting the stability of yeast strains containing multi-copy recombinant plasmids. Models for predicting plasmid loss are summarised, comparisons are made with relevant bacterial systems and strategies are described for overcoming such problems.     

New insights into cancer-related proteins provided by the yeast model

Cancer is a devastating disease with a profound impact on society. In recent years, yeast has provided a valuable contribution with respect to uncovering the molecular mechanisms underlying this disease, allowing the identification of new targets and novel therapeutic opportunities. Indeed, several attributes make yeast an ideal model system for the study of human diseases. It combines a high level of conservation between its cellular processes and those of mammalian cells, with advantages such as a short generation time, ease of genetic manipulation and a wealth of experimental tools for genome- and proteome-wide analyses. Additionally, the heterologous expression of disease-causing proteins in yeast has been successfully used to gain an understanding of the functions of these proteins and also to provide clues about the mechanisms of disease progression. Yeast research performed in recent years has demonstrated the tremendous potential of this model system, especially with the validation of findings obtained with yeast in more physiologically relevant models. The present review covers the major aspects of the most recent developments in the yeast research area with respect to cancer. It summarizes our current knowledge on yeast as a cellular model for investigating the molecular mechanisms of action of the major cancer-related proteins that, even without yeast orthologues, still recapitulate in yeast some of the key aspects of this cellular pathology. Moreover, the most recent contributions of yeast genetics and high-throughput screening technologies that aim to identify some of the potential causes underpinning this disorder, as well as discover new therapeutic agents, are discussed.     

High-throughput screening and selection of yeast cell lines expressing monoclonal antibodies

The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors.     

Overexpression of human calnexin in yeast improves measles surface glycoprotein solubility

The limitations of high-level expression of virus surface proteins in yeast are not well understood. The inefficiency of yeast to produce active human virus surface glycoproteins, as well as other mammalian glycoproteins, is usually explained by the inefficient folding of the glycoprotein into its characteristic and functional three-dimensional structure from a random coil. The endoplasmic reticulum (ER) is a highly versatile protein factory that is equipped with chaperones and folding enzymes essential for protein folding. To improve folding and solubility of viral surface glycoprotein, the genes encoding human ER resident chaperones calnexin, calreticulin, immunoglobin binding protein (BiP), protein disulfide isomerase (PDI) and foldase (ERp57) were coexpressed together with hemagglutinin gene from measles virus in the yeast Saccharomyces cerevisiae. The effect of coexpressing chaperones on the total yield of measles virus hemagglutinin (MeH) as well as the intracellular fate of the glycoprotein was determined. Our results demonstrated that coexpression of human calnexin noticeably enhanced the quantity of the soluble glycosylated form of MeH in yeast. The coexpression of human calreticulin-, PDI-, ERp57- and BiP-encoding genes did not improve the quality of recombinant MeH.     

Biosorption of cadmium ions by different yeast species

Toxicity and accumulation of Cd2+ in yeasts were studied in eight different yeast species. The adaptation to toxic concentration of this metal was dependent on the production of extracellular yeast glycoproteins. The highest concentration of Cd2+ ions in the growth medium was tolerated by a Hansenula anomala, strain while the lowest tolerance was found by the strain of species Saccharomyces cerevisiae. Extracellular glycoproteins of Hansenula anomala absorbed nearly 90% of the total content of Cd2+ ions bound by yeast cells, while extracellular glycoproteins of Saccharomyces cerevisiae bound only 6% of the total amount of cadmium. This difference is caused by the variable composition of the saccharide moiety in the extracellular glycoproteins. The composition of extracellular glycoproteins changed during the adaptation of the yeast cells to the presence of Cd2+ ions.     

Rtg2 protein: at the nexus of yeast longevity and aging

Firm support for the notion that metabolism and particularly mitochondrial metabolism plays a significant role in aging has been gathered in studies on yeast. As in other organisms, mitochondria contribute to aging through their propensity to generate reactive oxygen species. There is more to the involvement of mitochondria in aging than this, however. Mitochondrial dysfunction, which accumulates during aging, triggers the retrograde response, an intracellular signaling pathway that activates genes that compensate for this dysfunction. A key signaling protein in this pathway is the Rtg2 protein. Recent studies have provided evidence that this protein lies at the nexus of the four major processes that are involved in aging in yeast and in other organisms; namely, metabolism, stress resistance, chromatin-dependent gene regulation, and genome stability. The details of this central role of Rtg2 protein explain the delicate balance between longevity and aging, which ultimately must tip towards the latter. Phenomena that resemble the retrograde response appear to exist in human cells, with both common and cell type-specific gene expression changes as the output.     

Heterologous protein production in yeast

The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeastSaccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application.In the following review a selection from the different yeast systems is described and compared.     

Production of serpins using yeast expression systems

Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered.     

A combined yeast/bacteria two-hybrid system: development and evaluation

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.     

Production of recombinant proteins by yeast cells

Yeasts are widely used in production of recombinant proteins of medical or industrial interest. For each individual product, the most suitable expression system has to be identified and optimized, both on the genetic and fermentative level, by taking into account the properties of the product, the organism and the expression cassette. There is a wide range of important yeast expression hosts including the species Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Schizosaccharomyces pombe, Yarrowia lipolytica and Arxula adeninivorans, with various characteristics such as being thermo-tolerant or halo-tolerant, rapidly reaching high cell densities or utilizing unusual carbon sources. Several strains were also engineered to have further advantages, such as humanized glycosylation pathways or lack of proteases. Additionally, with a large variety of vectors, promoters and selection markers to choose from, combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology, it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins. In this review, the present status of the main and most promising yeast expression systems is discussed.     

Expression,glycosylation and secretion of fungal hydrolases in yeast

Although there are now some systems for transfer and expression of fungal genes, none gives expression at a level useful for production. There are useful expression systems in yeast, however, and these have been used to express genes coding for fungal extracellular hydrolases.This review examines how properties of the genes and gene product affect production and secretion of the enzyme in yeast. Similar considerations apply to expression of other heterologous genes in yeast and in other hosts.