Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins.

Surface display co-opts yeast's innate ability to embellish its cell wall with mannoproteins, thus converting the yeast's outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoform's N-terminus, e.g., through a short GPI-independent protein anchor. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the ß-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display of a reporter protein than the full-length Hsp150. Moreover, we developed a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display of a reporter protein as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of intracellular proteins into their N-terminally anchored isoforms.

Overexpressing CCW12 in Saccharomyces cerevisiae enables highly efficient ethanol production from lignocellulose hydrolysates.

A Saccharomyces cerevisiae strain CCW12OE was constructed by overexpressing CCW12 in a previously reported strain WXY70 harboring six xylose utilization genes. CCW12OE produced an optimal ethanol yield of 98.8% theoretical value within 48 h in a simulated corn stover hydrolysate. CCW12OEwas comprehensively evaluated for ethanol production in Miscanthus, maize and corncob hydrolysates, among which a 96.1% theoretical value was achieved within 12 h in corncob hydrolysates. Under normal growth conditions, CCW12OE did not display altered cell morphology; however, in the presence of acetate, CCW12OE maintained relatively intact cell structure and increased cell wall thickness by nearly 50%, while WXY70 had abnormal cell morphology and reduced cell wall thickness by nearly 50%. Besides, CCW12OE had higher fermentation capacity than that of WXY70 in undetoxified and detoxified hydrolysates with both aerobic and anaerobic conditions, demonstrating that CCW12 overexpression alone exhibits improved stress resistance and better fermentation performance.

Systematic Comparison of Cell Wall-Related Proteins of Different Yeasts.

Yeast cell walls have two major roles, to preserve physical integrity of the cell, and to ensure communication with surrounding molecules and cells. While the first function requires evolutionary conserved polysaccharide network synthesis, the second needs to be flexible and provide adaptability to different habitats and lifestyles. In this study, the comparative in silico analysis of proteins required for cell wall biosynthesis and functions containing 187 proteins of 92 different yeasts was performed in order to assess which proteins were broadly conserved among yeasts and which were more species specific. Proteins were divided into several groups according to their role and localization. As expected, many Saccharomyces cerevisiae proteins involved in protein glycosylation, glycosylphosphatidylinositol (GPI) synthesis and the synthesis of wall polysaccharides had orthologues in most other yeasts. Similarly, a group of GPI anchored proteins involved in cell wall biosynthesis (Gas proteins and Dfg5p/Dcw1p) and other non-GPI anchored cell wall proteins involved in the wall synthesis and remodeling were highly conserved. However, GPI anchored proteins involved in flocculation, aggregation, cell separation, and those of still unknown functions were not highly conserved. The proteins localized in the cell walls of various yeast species were also analyzed by protein biotinylation and blotting. Pronounced differences were found both in the patterns, as well as in the overall amounts of different groups of proteins. The amount of GPI-anchored proteins correlated with the mannan to glucan ratio of the wall. Changes of the wall proteome upon temperature shift to 42 °C were detected.

Evolutionary Overview of Molecular Interactions and Enzymatic Activities in the Yeast Cell Walls.

Fungal cell walls are composed of a polysaccharide network that serves as a scaffold in which different glycoproteins are embedded. Investigation of fungal cell walls, besides simple identification and characterization of the main cell wall building blocks, covers the pathways and regulations of synthesis of each individual component of the wall and biochemical reactions by which they are cross-linked and remodeled in response to different growth phase and environmental signals. In this review, a survey of composition and organization of so far identified and characterized cell wall components of different yeast genera including Saccharomyces, Candida, Kluyveromyces, Yarrowia, and Schizosaccharomyces are presented with the focus on their cell wall proteomes.

Comparison of two models of surface display of xylose reductase in the Saccharomyces cerevisiae cell wall.

In order to display xylose reductase at the surface of S. cerevisiae cells two different gene constructs have been prepared. In the first, xylose reductase gene GRE3 was fused with two parts of the CCW12 gene, the N-terminal one coding for the secretion signal sequence, and the C-terminal coding for the glycosylphosphatidylinositol anchoring signal. Transformed cells synthesized xylose reductase and incorporated it in the cell wall through the remnant of the glycosylphosphatidylinositol anchor. The other construct was prepared by fusing the GRE3 with the PIR4 gene coding for one of the proteins of the Pir-family containing the characteristic N-terminal repetitive sequence that anchors Pir proteins to ß-1,3-glucan. In this way xylose reductase was covalently attached to glucan through its N-terminus. For the expression of the constructs either the GAL1, or the PHO5 promoters have been used. Both strains displayed active xylose reductases and their enzyme properties were compared with the control enzyme bearing the secretion signal sequence but no anchoring signals, thus secreted into the medium. The enzyme displayed through the N-terminal fusion with PIR4 had higher affinity for xylose than the other construct, but they both expressed somewhat lower affinity than the control enzyme. Similarly, the Km values for NADPH of both immobilized enzymes were somewhat higher than the Km of the control XR. Both displayed enzymes, especially the one fused with Pir4, had higher thermal and pH stability than the control, while other enzymatic properties were not significantly impaired by surface immobilization.

Proteolytic processing of the Saccharomyces cerevisiae cell wall protein Scw4 regulates its activity and influences its covalent binding to glucan.

Yeast cell wall contains a number of proteins that are either non-covalently (Scw-proteins), or covalently (Ccw-proteins) bound to ß-1,3-glucan, the latter either through GPI-anchors and ß-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was shown that a part of Scw4, previously identified among the non-covalently bound cell wall proteins, was covalently attached to wall polysaccharides by a so far unknown alkali sensitive linkage. Thus Scw4 could be released from cell walls by treatments with hot SDS, mild alkali, or ß-1,3-glucanases, respectively. It was further shown that non-covalently bound Scw4 (SDS released) underwent the Kex2 proteolytic processing. In this paper it was demonstrated that Scw4 was also processed by yapsins at a position 9 amino acids downstream of the Kex2 cleavage site. Scw4 cleaved at the yapsin site had a markedly lower potential for covalent attachment to glucan. The overproduction of the fully processed form of Scw4 lead to high mortality, particularly in the stationary phase of growth, and to markedly increased cell size. On the other hand, the overproduction of Scw4 processed only by Kex2 or not processed at all had no apparent change in mortality indicating that only the smallest, completely mature form of Scw4 had the activity leading to observed phenotype changes.

Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd.

Purification and Characterization of a Novel Cold-Active Lipase from the Yeast Candida zeylanoides.

Cold-active lipases have attracted attention in recent years due to their potential applications in reactions requiring lower temperatures. Both bacterial and fungal lipases have been investigated, each having distinct advantages for particular applications. Among yeasts, cold-active lipases from the genera Candida, Yarrowia, Rhodotorula, and Pichia have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from Candida zeylanoides isolated from refrigerated poultry meat are described. Heat-sterilized olive oil was found to be the best lipase biosynthesis inducer, while nonionic detergents were not effective. The enzyme was purified to homogeneity using hydrophobic chromatography and its enzymatic properties were tested. Pure enzyme activity at 7 °C was about 60% of the maximal activity at 27 °C. The enzyme had rather good activity at higher temperatures, as well. Optimal pH of pure lipase was between 7.3 and 8.2, while the enzyme from the crude extract had an optimum pH of about 9.0. The enzyme was sensitive to high ionic strength and lost most of its activity at high salt concentrations. Due to the described properties, cold-active C. zeylanoides lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.

Proteins involved in building, maintaining and remodeling of yeast cell walls.

The cell wall defines the shape and provides osmotic stability to the yeast cell. It also serves to anchor proteins required for communication of the yeast cell with surrounding molecules and other cells. It is synthesized as a complex structure with ß-1,3-glucan chains forming the basic network to which ß-1,6-glucan, chitin and a number of mannoproteins are attached. Synthesis, maintaining and remodeling of this complex structure require a set of different synthases, hydrolases and transglycosidases whose concerted activities provide necessary firmness but at the same time flexibility of the wall moiety. The present state of comprehension of the interplay of these proteins in the yeast cell wall is the subject of this article.

Genetic immobilization of RNase Rny1p at the Saccharomyces cerevisiae cell surface.

Genetic immobilization of the yeast RNase Rny1p was performed by creating a hybrid protein containing the signal sequence of the S. cerevisiae cell wall protein Ccw12p followed by the catalytic part of the Rny1p (amino acids 19 to 293) and additionally 73 amino acids of the Ccw12p including the GPI-anchoring signal. The construct was expressed in S. cerevisiae VMY5678 and the hybrid protein was secreted through the plasma membrane and incorporated into the cell wall through GPI-anchoring in the same way as the Ccw12p. Thus, it could be released from the wall by ß-1,3-glucanase. It retained RNase activity with the optimal pH of about 9 and the optimal temperature at 60°C. It was significantly more stable than the wild type enzyme and retained activity at 50°C for at least 6 hours; at 60°C it maintained full activity for at least 4 h, and at 70°C it lost activity in about 2 h. No DNase activity of the Rny1/Ccw12p was detected. Yeast cells expressing the hybrid protein were successfully used instead of RNase A in a standard procedure for yeast chromosomal DNA preparation with the advantage of quick and easy quantitative removal of the RNase activity from the reaction mixture.

Correlation of liquid chromatographic and biological assay for potency assessment of filgrastim and related impurities.

In vivo and in vitro potency assays have always been a critical tool for confirmation of protein activity. However, due to their complexity and time consuming procedures, it remains a challenge to find an alternative analytical approach that would enable their replacement with no impact on the quality of provided information. The goal of this research was to determine if a correlation between liquid chromatography assays and in vitro biological assay could be established for filgrastim (recombinant human granulocyte-colony stimulating factor, rhG-CSF) samples containing various amounts of related impurities. For that purpose, relevant filgrastim related impurities were purified to homogeneity and characterized by liquid chromatography and mass spectrometry. A significant correlation (R(2)>0.90) between the two types of assays was revealed. Potency of oxidized filgrastim was determined to be approximately 25% of filgrastim stated potency (1 x 10(8)IU/mg of protein). Formyl-methionine filgrastim had potency of 89% of the filgrastim stated potency, while filgrastim dimer had 67% of filgrastim stated potency. A mathematical model for the estimation of biological activity of filgrastim samples from chromatography data was established and a significant correlation between experimental potency values and potency values estimated by the mathematical model was obtained (R(2)=0.92). Based on these results a conclusion was made that reversed phase high performance liquid chromatography could be used as an alternative for the in vitro biological assay for potency assessment of filgrastim samples. Such an alternative model would enable substitution of a complex and time consuming biological assay with a robust and precise instrumental method in many practical cases.