Steven Geysens

Bio: Steven Geysens is an academic researcher from Ghent University. The author has contributed to research in topics: Glycosylation & Pichia pastoris. The author has an hindex of 16, co-authored 32 publications receiving 2365 citations.

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

Abstract: The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Engineering complex-type N-glycosylation in Pichia pastoris using GlycoSwitch technology

Abstract: Here we provide a protocol for engineering the N-glycosylation pathway of the yeast Pichia pastoris. The general strategy consists of the disruption of an endogenous glycosyltransferase gene (OCH1) and the stepwise introduction of heterologous glycosylation enzymes. Each engineering step results in the introduction of one glycosidase or glycosyltransferase activity into the Pichia endoplasmic reticulum or Golgi complex and consists of a number of stages: transformation with the appropriate GlycoSwitch vector, small-scale cultivation of a number of transformants, sugar analysis and heterologous protein expression analysis. If desired, the resulting clone can be further engineered by repeating the procedure with the next GlycoSwitch vector. Each engineering step takes approximately 3 weeks. The conversion of any wild-type Pichia strain into a strain that modifies its glycoproteins with Gal(2)GlcNAc(2)Man(3)GlcNAc(2)N-glycans requires the introduction of five GlycoSwitch vectors. Three examples of the full engineering procedure are provided to illustrate the results that can be expected.

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris

Abstract: The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.

Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment

Abstract: The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.

Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A

Abstract: The glycosylation of Cel7A (CBH I) from Trichoderma reeseivaries considerably when the fungus is grown under differentconditions. As shown by ESI-MS and PAG-IEF analyses ofboth intact protein and the isolated catalytic core module, themicroheterogeneity originates mainly from the variable ratioof single N-acetylglucosamine over high-mannose structureson the three N-glycosylation sites and from the presence orabsence of phosphate residues. Fully N- and O-glycosylatedCel7A can only be isolated from minimal medium and prob-ablyreflectstheinitialcomplexityoftheproteinonleavingtheglycosynthetic pathway. Extracellular activities are responsi-ble for postsecretorial modifications in other cultivation con-ditions: a-(1!2)-mannosidase, a-(1!3)-glucosidase and anEndo H type activity participate in N-deglycosylation (core),whereas a phosphatase and a mannosidase are probablyresponsible for hydrolysis of O-glycans (linker). The effectsare most prominent in corn steep liquor–enriched media,where the pH is closer to the pH optimum (5–6) of theseextracellular hydrolases. In minimal medium, the low pH andthepresenceofproteasescouldexplainfortheabsenceofsuchactivities. On the other hand, phosphodiester linkages in thecatalytic module are only observed under specific conditions.The extracellular trigger is still unknown, but manno-phosphorylationmayberegulatedintracellularlybya-(1!2)-mannosidases and phosphomannosyl transferases competingfor the same intermediate in the glycosynthetic pathway.Key words: Cel7A/endoglycosidase/N- andO-glycosylation/postsecretorial modifications/Trichoderma reeseiIntroductionCellulases belonging to different glycosyl hydrolase familiesvery often exhibit a multidomain structure (Coutinhoand Henrissat, 1999): a catalytic domain (core) and acarbohydrate-binding module are separated by a linkerpeptide rich in proline, serine, and threonine. Fungalcellulases carry posttranslational modifications on bothdomains: Whereas the linker peptide is highly O-glycosylated, N-glycosylation seems to be restricted to thecore. Filamentous fungi typically synthesize short-chain O-and N-glycans, resembling the mammalian high-mannosetype rather than the hyperglycosylated yeast structures.The glycosylation of cellobiohydrolase I (CBH I, Cel7A),a cellulase abundantly expressed by most Trichodermareesei strains, has been studied particularly well(Table I). Trichoderma cellulases appear in several isoformswith similar catalytic and adsorption properties (Medveet al., 1998), and it has been shown that both N- andO-glycans account for the many isoforms of Cel7A(Pakula et al., 2000).Detailed structural investigations (Harrison et al., 1998;Klarskov et al., 1997) revealed the N-glycosylation inT. reesei Cel7A from respectively strains QM9414 andALKO2877 (derived from QM9414): Single GlcNAc resi-dues in three (Asn45, Asn270, and Asn384) out of fourpotential sites were characterized in the catalytic domain.More complex N-glycan structures were observed withCel7A from the hyperproducing mutant Rut-C30 strain(Maras et al., 1997). The majority are monoglucosylatedhigh-mannose glycans (GlcMan

Heterologous protein production using the Pichia pastoris expression system.

Abstract: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina).

Abstract: Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.

Deconstruction of lignocellulosic biomass to fuels and chemicals

Abstract: Plants represent a vast, renewable resource and are well suited to provide sustainably for humankind’s transportation fuel needs. To produce infrastructure-compatible fuels from biomass, two challenges remain: overcoming plant cell wall recalcitrance to extract sugar and phenolic intermediates, and reduction of oxygenated intermediates to fuel molecules. To compete with fossil-based fuels, two primary routes to deconstruct cell walls are under development, namely biochemical and thermochemical conversion. Here, we focus on overcoming recalcitrance with biochemical conversion, which uses low-severity thermochemical pretreatment followed by enzymatic hydrolysis to produce soluble sugars. Many challenges remain, including understanding how pretreatments affect the physicochemical nature of heterogeneous cell walls; determination of how enzymes deconstruct the cell wall effectively with the aim of designing superior catalysts; and resolution of issues associated with the co-optimization of pretreatment, enzymatic hydrolysis, and fermentation. Here, we highlight some of the scientific challenges and open questions with a particular focus on problems across multiple length scales.

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.

Abstract: Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.