Mannosylated hemagglutinin peptides bind cyanovirin-N independent of disulfide-bonds in complementary binding sites.

Cyanovirin-N (CV-N) has been shown to reveal broad neutralizing activity against human immunodeficiency virus (HIV) and to specifically bind Manα(1→2)Manα units exposed on various glycoproteins of enveloped viruses, such as influenza hemagglutinin (HA) and Ebola glycoprotein. Chemically synthesized dimannosylated HA peptides bound domain-swapped and dimeric CV-N with either four disulfide-bonds (Cys-Cys), or three Cys-Cys bonds and an intact fold of the high-affinity binding site at an equilibrium dissociation constant of 10 μM. Cys-Cys mutagenesis with ion-pairing amino-acids glutamic acid and arginine was calculated by structure-based protein design and allowed for recognizing dimannose and dimannosylated peptide binding to low-affinity binding sites ( ≈ 11 μM for one C58-C73 bond, and binding to dimannosylated peptide).

The adaptive landscape of wildtype and glycosylation-deficient populations of the industrial yeast Pichia pastoris.

Background: The effects of long-term environmental adaptation and the implications of major cellular malfunctions are still poorly understood for non-model but biotechnologically relevant species. In this study we performed a large-scale laboratory evolution experiment with 48 populations of the yeast Pichia pastoris in order to establish a general adaptive landscape upon long-term selection in several glucose-based growth environments. As a model for a cellular malfunction the implications of OCH1 mannosyltransferase knockout-mediated glycosylation-deficiency were analyzed.

Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris.

Background: Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P.

The underestimated N-glycomes of lepidopteran species.

Background: Insects are significant to the environment, agriculture, health and biotechnology. Many of these aspects display some relationship to glycosylation, e.g.

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans.

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that "simple" organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N'-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain.

Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases.

Fused lobes (FDL) hexosaminidases are the most recently genetically defined glycosidases involved in the biosynthesis of N-glycans in invertebrates, and their narrow specificity is essential for the generation of paucimannosidic N-glycans in insects. In this study, we explored the potential of FDL hexosaminidases in the utilization of different artificial and natural substrates, both as purified, native compounds or generated in vitro using various relevant glycosyltransferases. In addition to the already-known FDL enzyme from Drosophila melanogaster, we now have identified and characterized the Apis mellifera FDL homolog.

N-glycomic profiling of a glucosidase II mutant of Dictyostelium discoideum by ''off-line'' liquid chromatography and mass spectrometry.

In this study, we have performed the first mass spectrometric analysis of N-glycans of the M31 mutant strain of the cellular slime mould Dictyostelium discoideum, previously shown to have a defect in glucosidase II. Together with glucosidase I, this enzyme mediates part of the initial processing of N-glycans; defects in either glucosidase are associated with human diseases and result in an accumulation of incorrectly processed oligosaccharides which are not, or only poor, substrates for a range of downstream enzymes. To examine the effect of the glucosidase II mutation in Dictyostelium, we employed off-line LC-MALDI-TOF MS in combination with chemical and enzymatic treatments and MS/MS to analyze the neutral and anionic N-glycans of the mutant as compared to the wild type.

Recombinant Aspergillus β-galactosidases as a robust glycomic and biotechnological tool.

Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized β-galactosidases from Aspergillus nidulans as well as one β-galactosidase from Aspergillus niger.

Adaptive laboratory evolution -- principles and applications for biotechnology.

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations.

Evolutionary potential, cross-stress behavior and the genetic basis of acquired stress resistance in Escherichia coli.

Bacterial populations have a remarkable capacity to cope with extreme environmental fluctuations in their natural environments. In certain cases, adaptation to one stressful environment provides a fitness advantage when cells are exposed to a second stressor, a phenomenon that has been coined as cross-stress protection. A tantalizing question in bacterial physiology is how the cross-stress behavior emerges during evolutionary adaptation and what the genetic basis of acquired stress resistance is.

A synthetic biology approach to self-regulatory recombinant protein production in Escherichia coli.

Background: Recombinant protein production is a process of great industrial interest, with products that range from pharmaceuticals to biofuels. Since high level production of recombinant protein imposes significant stress in the host organism, several methods have been developed over the years to optimize protein production. So far, these trial-and-error techniques have proved laborious and sensitive to process parameters, while there has been no attempt to address the problem by applying Synthetic Biology principles and methods, such as integration of standardized parts in novel synthetic circuits.

Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth.

The demand for recombinant proteins both for biopharmaceutical and technical applications is rapidly growing, and therefore the need to establish highly productive expression systems is steadily increasing. Yeasts, such as Pichia pastoris, are among the widely used production platforms with a strong emphasis on secreted proteins. Protein secretion is a limiting factor of productivity.

The impact of oxygen on the transcriptome of recombinant S. cerevisiae and P. pastoris - a comparative analysis.

Background: Saccharomyces cerevisiae and Pichia pastoris are two of the most relevant microbial eukaryotic platforms for the production of recombinant proteins. Their known genome sequences enabled several transcriptomic profiling studies under many different environmental conditions, thus mimicking not only perturbations and adaptations which occur in their natural surroundings, but also in industrial processes. Notably, the majority of such transcriptome analyses were performed using non-engineered strains.

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis.

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance.

Induction and measurement of UPR and osmotic stress in the yeast Pichia pastoris.

Unfolded protein response (UPR) is a major reaction to intrinsic stress of eukaryotic organisms and is also related to environmental stress reactions. Among yeasts, stress regulation has mainly been investigated in Saccharomyces cerevisiae, while other species with biotechnological or medical interest are less well understood. Pichia pastoris as one example has emerged as a favorite production platform for recombinant proteins during the last two decades.

A multi-level study of recombinant Pichia pastoris in different oxygen conditions.

Background: Yeasts are attractive expression platforms for many recombinant proteins, and there is evidence for an important interrelation between the protein secretion machinery and environmental stresses. While adaptive responses to such stresses are extensively studied in Saccharomyces cerevisiae, little is known about their impact on the physiology of Pichia pastoris. We have recently reported a beneficial effect of hypoxia on recombinant Fab secretion in P.

Engineering of biotin-prototrophy in Pichia pastoris for robust production processes.

Biotin plays an essential role as cofactor for biotin-dependent carboxylases involved in essential metabolic pathways. The cultivation of Pichia pastoris, a methylotrophic yeast that is successfully used as host for the production of recombinant proteins, requires addition of high dosage of biotin. As biotin is the only non-salt media component used during P.

The response to unfolded protein is involved in osmotolerance of Pichia pastoris.

Background: The effect of osmolarity on cellular physiology has been subject of investigation in many different species. High osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. Several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms.

Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris.

Background: Pichia pastoris is widely used as a production platform for heterologous proteins and model organism for organelle proliferation. Without a published genome sequence available, strain and process development relied mainly on analogies to other, well studied yeasts like Saccharomyces cerevisiae.

Results: To investigate specific features of growth and protein secretion, we have sequenced the 9.

Yeast systems biotechnology for the production of heterologous proteins.

Systems biotechnology has been established as a highly potent tool for bioprocess development in recent years. The applicability to complex metabolic processes such as protein synthesis and secretion, however, is still in its infancy. While yeasts are frequently applied for heterologous protein production, more progress in this field has been achieved for bacterial and mammalian cell culture systems than for yeasts.

The effect of temperature on the proteome of recombinant Pichia pastoris.

The impact of environmental factors on the productivity of yeast cells is poorly investigated so far. Therefore, it is a major concern to improve the understanding of cellular physiology of microbial protein production hosts, including the methylotrophic yeast Pichia pastoris. Two-Dimensional Fluorescence Difference Gel electrophoresis and protein identification via mass spectrometry were applied to analyze the impact of cultivation temperature on the physiology of a heterologous protein secreting P.

Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays.

Background: DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae.

Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview.

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells.

Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins.

High cell density cultivation of Pichia pastoris has to cope with several technical limitations, most importantly the transfer of oxygen. By applying hypoxic conditions to chemostat cultivations of P. pastoris expressing an antibody Fab fragment under the GAP promoter, a 2.