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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 K D of 10 μM. Cys-Cys mutagenesis with ion-pairing amino-acids glutamic acid and arginine was calculated by in silico structure-based protein design and allowed for recognizing dimannose and dimannosylated peptide binding to low-affinity binding sites (K D ≈ 11 μM for one C58-C73 bond, and binding to dimannosylated peptide). In comparison, binding to HA was achieved based on one ion-pairing C58E-C73R substitution at K D = 275 nM, and K D = 5 μM for two C58E-C73R substitutions. We were utilizing a triazole bioisostere linkage to form the respective mannosylated-derivative on the HA peptide sequence of residues glutamine, glycine, and glutamic acid. Thus, mono- and dimannosylated peptides with N-terminal cysteine facilitated site-specific interactions with HA peptides, mimicking a naturally found N-linked glycosylation site on the HA head domain.

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. To address these questions, we evolved Escherichia coli cells over 500 generations in five environments that include four abiotic stressors. Through growth profiling and competition assays, we identified several cases of positive and negative cross-stress behavior that span all strain-stress combinations. Resequencing the genomes of the evolved strains resulted in the identification of several mutations and gene amplifications, whose fitness effect was further assessed by mutation reversal and competition assays. Transcriptional profiling of all strains under a specific stress, NaCl-induced osmotic stress, and integration with resequencing data further elucidated the regulatory responses and genes that are involved in this phenomenon. Our results suggest that cross-stress dependencies are ubiquitous, highly interconnected, and can emerge within short timeframes. The high adaptive potential that we observed argues that bacterial populations occupy a genotypic space that enables a high phenotypic plasticity during adaptation in fluctuating environments.

Abstract 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. Results We present a novel self-regulatory protein production system that couples the control of recombinant protein production with a stress-induced, negative feedback mechanism. The synthetic circuit allows the down-regulation of recombinant protein expression through a stress-induced promoter. We used E. coli as the host organism, since it is widely used in recombinant processes. Our results show that the introduction of the self-regulatory circuit increases the soluble/insoluble ratio of recombinant protein at the expense of total protein yield. To further elucidate the dynamics of the system, we developed a computational model that is in agreement with the observed experimental data, and provides insight on the interplay between protein solubility and yield. Conclusion Our work introduces the idea of a self-regulatory circuit for recombinant protein products, and paves the way for processes with reduced external control or monitoring needs. It demonstrates that the library of standard biological parts serves as a valuable resource for initial synthetic blocks that needs to be further refined to be successfully applied in practical problems of biotechnological significance. Finally, the development of a predictive model in conjunction with experimental validation facilitates a better understanding of the underlying dynamics and can be used as a guide to experimental design.

Abstract 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. In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. Results The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. Conclusions The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Abstract 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. In this comparative study, the gene expression profiles of S. cerevisiae and P. pastoris, a Crabtree positive and Crabtree negative yeast, respectively, were analyzed for three different oxygenation conditions (normoxic, oxygen-limited and hypoxic) under recombinant protein producing conditions in chemostat cultivations. Results The major differences in the transcriptomes of S. cerevisiae and P. pastoris were observed between hypoxic and normoxic conditions, where the availability of oxygen strongly affected ergosterol biosynthesis, central carbon metabolism and stress responses, particularly the unfolded protein response. Steady state conditions under low oxygen set-points seemed to perturb the transcriptome of S. cerevisiae to a much lesser extent than the one of P. pastoris, reflecting the major tolerance of the baker's yeast towards oxygen limitation, and a higher fermentative capacity. Further important differences were related to Fab production, which was not significantly affected by oxygen availability in S. cerevisiae, while a clear productivity increase had been previously reported for hypoxically grown P. pastoris. Conclusions The effect of three different levels of oxygen availability on the physiology of P. pastoris and S. cerevisiae revealed a very distinct remodelling of the transcriptional program, leading to novel insights into the different adaptive responses of Crabtree negative and positive yeasts to oxygen availability. Moreover, the application of such comparative genomic studies to recombinant hosts grown in different environments might lead to the identification of key factors for efficient protein production.

Abstract 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. Thus, the effect of osmolarity on the cellular physiology of Pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. Transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. While our main intention was to analyze the effect of different osmolarities on P. pastoris in general, this was complemented by studying it in context with recombinant protein production. Results In contrast to the model yeast Saccharomyces cerevisiae, the main osmolyte in P. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2D Fluorescence Difference Gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. These data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a P. pastoris strain, secreting the Fab fragment, compared with the wild type strain. No transcriptional activation of the high osmolarity glycerol (HOG) pathway was observed at steady state conditions. Furthermore, no change of the specific productivity of recombinant Fab was observed at increased osmolarity. Conclusion These data point out that the physiological response to increased osmolarity is different to S. cerevisiae. Increased osmolarity resulted in an unfolded protein response (UPR) like response in P. pastoris and lead to pre-conditioning of the recombinant Fab producing strain of P. pastoris to growth at high osmolarity. The current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. Therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast.

Abstract 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 9.4 Mb genome of the type strain DSMZ 70382 and analyzed the secretome and the sugar transporters. The computationally predicted secretome consists of 88 ORFs. When grown on glucose, only 20 proteins were actually secreted at detectable levels. These data highlight one major feature of P. pastoris, namely the low contamination of heterologous proteins with host cell protein, when applying glucose based expression systems. Putative sugar transporters were identified and compared to those of related yeast species. The genome comprises 2 homologs to S. cerevisiae low affinity transporters and 2 to high affinity transporters of other Crabtree negative yeasts. Contrary to other yeasts, P. pastoris possesses 4 H+/glycerol transporters. Conclusion This work highlights significant advantages of using the P. pastoris system with glucose based expression and fermentation strategies. As only few proteins and no proteases are actually secreted on glucose, it becomes evident that cell lysis is the relevant cause of proteolytic degradation of secreted proteins. The endowment with hexose transporters, dominantly of the high affinity type, limits glucose uptake rates and thus overflow metabolism as observed in S. cerevisiae. The presence of 4 genes for glycerol transporters explains the high specific growth rates on this substrate and underlines the suitability of a glycerol/glucose based fermentation strategy. Furthermore, we present an open access web based genome browser http://www.pichiagenome.org.

Abstract 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. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results.

Abstract 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. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.