Production of recombinant proteins by filamentous fungi

The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientific information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be confined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology.     

ATG genes involved in non-selective autophagy are conserved from yeast to man, but the selective Cvt and pexophagy pathways also require organism-specific genes

ATG genes encode proteins that are required for macroautophagy, the Cvt pathway and/or pexophagy. Using the published Atg protein sequences, we have screened protein and DNA databases to identify putative functional homologs (orthologs) in 21 fungal species (yeast and filamentous fungi) of which the genome sequences were available. For comparison with Atg proteins in higher eukaryotes, also an analysis of Arabidopsis thaliana and Homo sapiens databases was included. This analysis demonstrated that Atg proteins required for non-selective macroautophagy are conserved from yeast to man, stressing the importance of this process in cell survival and viability. The A. thaliana and human genomes encode multiple proteins highly similar to specific fungal Atg proteins (paralogs), possibly representing cell type-specific isoforms. The Atg proteins specifically involved in the Cvt pathway and/or pexophagy showed poor conservation, and were generally not present in A. thaliana and man. Furthermore, Atg19, the receptor of Cvt cargo, was only detected in Saccharomyces cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, the Cvt complex) to the autophagic machinery was identified in all yeast species and filamentous fungi under study. This suggests that in fungi an organism-specific form of selective autophagy may occur, for which specialized Atg proteins have evolved.     

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'top-down' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.     

Filamentous fungi as production organisms for glycoproteins of bio-medical interest

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20–40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but research is in progress to increase the production levels. In the past years the structure of protein-linked carbohydrates of a number of fungal proteins has been elucidated, showing the presence of oligo-mannosidic and high-mannose chains, sometimes with typical fungal modifications. A start has been made to engineer the glycosylation pathway in filamentous fungi to obtain strains that show a more mammalian-like type of glycosylation. This mini review aims to cover the current knowledge of glycosylation in filamentous fungi, and to show the possibilities to produce glycoproteins with these organisms with a more mammalian-like type of glycosylation for research purposes or pharmaceutical applications     

Engineering of glycosylation in yeast and other fungi: current state and perspectives

With the increasing demand for recombinant proteins and glycoproteins, research on hosts for producing these proteins is focusing increasingly on more cost-effective expression systems. Yeasts and other fungi are promising alternatives because they provide easy and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and other fungi modify their glycoproteins with heterogeneous high-mannose glycan structures, which is often detrimental to a therapeutic protein’s pharmacokinetic behavior and can reduce the efficiency of downstream processing. This problem can be solved by engineering the glycosylation pathways to produce homogeneous and, if so desired, human-like glycan structures. In this review, we provide an overview of the most significant recently reported approaches for engineering the glycosylation pathways in yeasts and fungi.     

Role of N- and O-glycans in polarized biosynthetic sorting

The maintenance of proper epithelial function requires efficient sorting of newly synthesized and recycling proteins to the apical and basolateral surfaces of differentiated cells. Whereas basolateral protein sorting signals are generally confined to their cytoplasmic regions, apical targeting signals have been identified that localize to luminal, transmembrane, and cytoplasmic aspects of proteins. In the past few years, both N- and O-linked glycans have been identified as apical sorting determinants. Glycan structures are extraordinarily diverse and have tremendous information potential. Moreover, because the oligosaccharides added to a given protein can change depending on cell type and developmental stage, the potential exists for altering sorting pathways by modulation of the expression pattern of enzymes involved in glycan synthesis. In this review, we discuss the evidence for glycan-mediated apical sorting along the biosynthetic pathway and present possible mechanisms by which these common and heterogeneous posttranslational modifications might function as specific sorting signals. glycosylation; epithelia; polarity; kidney; intestine     

Aquaporins in yeasts and filamentous fungi

Recently, genome sequences from different fungi have become available. This information reveals that yeasts and filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budding yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. Whereas Aqy1 is a spore-specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmotic signals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the otherwise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1-like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, plays a central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gating have been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has a long N-terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins, only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi are a fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compare orthologous sequences from a large number of different organisms facilitates functional and structural studies.     

Signal transduction cascades regulating fungal development and virulence.

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.     

Signal Transduction Cascades Regulating Fungal Development and Virulence

Cellular differentiation, mating, and filamentous growth are regulated in many fungi by environmental and nutritional signals. For example, in response to nitrogen limitation, diploid cells of the yeast Saccharomyces cerevisiae undergo a dimorphic transition to filamentous growth referred to as pseudohyphal differentiation. Yeast filamentous growth is regulated, in part, by two conserved signal transduction cascades: a mitogen-activated protein kinase cascade and a G-protein regulated cyclic AMP signaling pathway. Related signaling cascades play an analogous role in regulating mating and virulence in the plant fungal pathogen Ustilago maydis and the human fungal pathogens Cryptococcus neoformans and Candida albicans. We review here studies on the signaling cascades that regulate development of these and other fungi. This analysis illustrates both how the model yeast S. cerevisiae can serve as a paradigm for signaling in other organisms and also how studies in other fungi provide insights into conserved signaling pathways that operate in many divergent organisms.     

N-Glycan Modification in Aspergillus Species

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man₅GlcNAc₂ type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan₅GlcNac₂ N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man₃GlcNAc₂ N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.     

Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN.     

Mass spectrometry for congenital disorders of glycosylation, CDG

Congenital disorders of glycosylation (CDG) constitute a group of diseases affecting N-linked glycosylation pathways. The classical type of CDG, now called CDG-I, results from deficiencies in the early glycosylation pathway for biosynthesis of lipid-linked oligosaccharide and its transfer to proteins in endoplasmic reticulum, while the CDG-II diseases are caused by defects in the subsequent processing steps. Mass spectrometry (MS) produced a milestone in CDG research, by localizing the CDG-I defect to the early glycosylation pathway in 1992. Currently, MS of transferrin, either by electrospray ionization or matrix-assisted laser desorption/ionization, plays the central role in laboratory screening of CDG-I. On the other hand, the glycopeptide analysis recently developed for site-specific glycans of glycoproteins allows detailed glycan analysis in a high throughput manner and will solve problems in CDG-II diagnosis. These techniques will facilitate studying CDG, a field now expanding to O-linked glycosylation and to acquired as well as inherited conditions that can affect protein glycosylation.     

Protein glycosylation in bacterial mucosal pathogens

In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events that are required for biological processes ranging from immune recognition to cancer development. Glycosylation was previously considered to be restricted to eukaryotes; however, through advances in analytical methods and genome sequencing, there have been increasing reports of both O-linked and N-linked protein glycosylation pathways in bacteria, particularly amongst mucosal-associated pathogens. Studying glycosylation in relatively less-complicated bacterial systems provides the opportunity to elucidate and exploit glycoprotein biosynthetic pathways. We will review the genetic organization, glycan structures and function of glycosylation systems in mucosal bacterial pathogens, and speculate on how this knowledge may help us to understand glycosylation processes in more complex eukaryotic systems and how it can be used for glycoengineering.     

Harnessing glycosylation to improve cellulase activity

Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impact enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.     

Characterization of the α-mannosidase gene family in filamentous fungi: N-glycan remodelling for the development of eukaryotic expression systems

Although filamentous fungi are used extensively for protein expression, their use for the production of heterologous glycoproteins is constrained by the types of N-glycan structures produced by filamentous fungi as compared to those naturally found on the glycoproteins. Attempts are underway to engineer the N-glycan synthetic pathways in filamentous fungi in order to produce fungal expression strains which can produce heterologous glycoproteins carrying specific N-glycan structures. To fully realize this goal, a detailed understanding of the genetic components of this pathway in filamentous fungi is required. In this review, we discuss the characterization of the α-mannosidase gene family in filamentous fungi and its implications for the elucidation of the N-glycan synthetic pathway.     

Conserved processes and lineage-specific proteins in fungal cell wall evolution

The cell wall is a defining organelle that differentiates fungi from its sister clades in the opisthokont superkingdom. With a sensitive technique to align low-complexity protein sequences, we have identified 187 cell wall-related proteins in Saccharomyces cerevisiae and determined the presence or absence of homologs in 17 other fungal genomes. There were both conserved and lineage-specific cell wall proteins, and the degree of conservation was strongly correlated with protein function. Some functional classes were poorly conserved and lineage specific: adhesins, structural wall glycoprotein components, and unannotated open reading frames. These proteins are primarily those that are constituents of the walls themselves. On the other hand, glycosyl hydrolases and transferases, proteases, lipases, proteins in the glycosyl phosphatidyl-inositol-protein synthesis pathway, and chaperones were strongly conserved. Many of these proteins are also conserved in other eukaryotes and are associated with wall synthesis in plants. This gene conservation, along with known similarities in wall architecture, implies that the basic architecture of fungal walls is ancestral to the divergence of the ascomycetes and basidiomycetes. The contrasting lineage specificity of wall resident proteins implies diversification. Therefore, fungal cell walls consist of rapidly diversifying proteins that are assembled by the products of an ancestral and conserved set of genes.     

Biochemical characterization of the O-linked glycosylation pathway in Neisseria gonorrhoeae responsible for biosynthesis of protein glycans containing N,N'-diacetylbacillosamine

The O-linked protein glycosylation pathway in Neisseria gonorrhoeae is responsible for the synthesis of a complex oligosaccharide on undecaprenyl diphosphate and subsequent en bloc transfer of the glycan to serine residues of select periplasmic proteins. Protein glycosylation (pgl) genes have been annotated on the basis of bioinformatics and top-down mass spectrometry analysis of protein modifications in pgl-null strains [Aas, F. E., et al. (2007) Mol. Microbiol. 65, 607-624; Vik, A., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 4447-4452], but relatively little biochemical analysis has been performed to date. In this report, we present the expression, purification, and functional characterization of seven Pgl enzymes. Specifically, the enzymes studied are responsible for synthesis of an uncommon uridine diphosphate (UDP)-sugar (PglD, PglC, and PglB-acetyltransferase domain), glycan assembly (PglB-phospho-glycosyltransferase domain, PglA, PglE, and PglH), and final oligosaccharide transfer (PglO). UDP-2,4-diacetamido-2,4,6-trideoxy-α-d-hexose (DATDH), which is the first sugar in glycan biosynthesis, was produced enzymatically, and the stereochemistry was assigned as uridine diphosphate N'-diacetylbacillosamine (UDP-diNAcBac) by nuclear magnetic resonance characterization. In addition, the substrate specificities of the phospho-glycosyltransferase, glycosyltransferases, and oligosaccharyltransferase (OTase) were analyzed in vitro, and in most cases, these enzymes exhibited strong preferences for the native substrates relative to closely related glycans. In particular, PglO, the O-linked OTase, and PglB(Cj), the N-linked OTase from Campylobacter jejuni, preferred the native N. gonorrhoeae and C. jejuni substrates, respectively. This study represents the first comprehensive biochemical characterization of this important O-linked glycosylation pathway and provides the basis for further investigations of these enzymes as antibacterial targets.     

Structure and synthesis of polyisoprenoids used in N-glycosylation across the three domains of life

N-linked protein glycosylation was originally thought to be specific to eukaryotes, but evidence of this post-translational modification has now been discovered across all domains of life: Eucarya, Bacteria, and Archaea. In all cases, the glycans are first assembled in a step-wise manner on a polyisoprenoid carrier lipid. At some stage of lipid-linked oligosaccharide synthesis, the glycan is flipped across a membrane. Subsequently, the completed glycan is transferred to specific asparagine residues on the protein of interest. Interestingly, though the N-glycosylation pathway seems to be conserved, the biosynthetic pathways of the polyisoprenoid carriers, the specific structures of the carriers, and the glycan residues added to the carriers vary widely. In this review we will elucidate how organisms in each basic domain of life synthesize the polyisoprenoids that they utilize for N-linked glycosylation and briefly discuss the subsequent modifications of the lipid to generate a lipid-linked oligosaccharide.