Cell-surface expression of Aspergillus saitoi-derived functional α-1,2-mannosidase on Yarrowia lipolytica for glycan remodeling

Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man₈GlcNAc₂ to Man₅GlcNAc₂ efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.     

Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum)

Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves.     

p24 proteins play a role in peroxisome proliferation in yeast

Emp24 is a member of the p24 protein family, which was initially localized to the endoplasmic reticulum, Golgi and COP vesicles, but has recently shown to be associated with Saccharomyces cerevisiae peroxisomes as well. Using cell fractionation and electron- and fluorescence microscopy, we show that in the yeast Hansenula polymorpha, Emp24 also associates with peroxisomes. In addition, we show that peroxisome numbers are strongly decreased in H. polymorpha cells lacking two proteins of the p24 complex, Emp24 and Erp3. Detailed fluorescence microscopy analyses suggest that emp24.erp3 cells are disturbed in peroxisome fission and inheritance.     

Scarless gene deletion in methylotrophic Hansenula polymorpha by using mazF as counter-selectable marker

With increasing application of Hansenula polymorpha in fundamental research and biotechnology, many more genetic manipulations are required. However, these have been restricted for the finiteness of selectable markers. Here, MazF, a toxin protein from Escherichia coli, was investigated as a counter-selectable marker in H. polymorpha. The lethal effect of MazF on yeast cells suggested that it is a candidate for counter-selection in H. polymorpha. Markerless or scarless gene deletion in H. polymorpha was conducted based on selectable markers cassette mazF-zeoR, in which the zeocin resistance cassette and mazF expression cassette were used as positive and counter-selectable markers, respectively. For markerless deletion, the target region can be replaced by CYC1TT via two-step homologous recombination. For scarless deletion, the innate upstream region (5′UP) of target genes rather than CYC1TT mediates homologous recombination to excise both selectable markers and 5′ sequence of target genes. Moreover, scarless deletion can be accomplished by using short homologous arms for the effectiveness of mazF as a counter-selectable marker. The applicability of the strategies in markerless or scarless deletion of PEP4, LEU2, and TRP1 indicates that this study provides easy, time-efficient, and host-independent protocols for single or multiple genetic manipulations in H. polymorpha.     

Chromosome Polymorphisms among Strains of Hansenula polymorpha (syn. Pichia angusta)

Contour-clamped homogeneous electrophoresis and an embedded-agarose method of sample preparation were combined to carry out an analysis of the chromosome sets of nine strains of Hansenula polymorpha (syn. Pichia angusta). Chromosomal DNA molecules could be separated into a series of bands ranging, approximately, from 650 up to 2,200 kb in size. Polymorphism of the electrophoretic pattern was demonstrated among the strains investigated in this study. Cross-hybridization between H. polymorpha and Saccharomyces cerevisiae ribosomal DNA was also observed.     

Possible Association of GroES and Antigen 85 Proteins with Heat Resistance of Mycobacterium paratuberculosis

Conflicting reports on the heat resistance of Mycobacterium paratuberculosis prompted an examination of the effect of culture medium on this property of the organism. M. paratuberculosis was cultured in three types of media (fatty acid-containing medium 7H9-OADC (oleic acid-albumin-dextrose-catalase supplement) and glycerol-containing media WR-GD and 7H9-GD [glycerol-dextrose supplement]) at pH 6.0. M. paratuberculosis grown under these three culture conditions was then tested for heat resistance in distilled water at 65°C. Soluble proteins and mycolic acids of M. paratuberculosis were evaluated by two-dimensional electrophoresis (2-DE) and thin-layer chromatography (TLC), respectively. The type of culture medium used significantly affected the heat resistance of M. paratuberculosis. The decimal reduction times at 65°C (D_65°C values; times required to reduce the concentration of bacteria by a factor of 10 at 65°C) for M. paratuberculosis strains grown in 7H9-OADC were significantly higher than those for the organisms grown in WR-GD medium (P < 0.01). When the glycerol-dextrose supplement of WR was substituted for the fatty acid supplement (OADC) in 7H9 medium (resulting in 7H9-GD), the D_65°C value was significantly lower than that for the organism grown in 7H9-OADC medium (P = 0.022) but higher than that when it was cultured in WR-GD medium (P = 0.005). Proteomic analysis by 2-DE of soluble proteins extracted from M. paratuberculosis grown without heat stress in the three media (7H9-OADC, 7H9-GD, and WR-GD) revealed that seven proteins were more highly expressed in 7H9-OADC medium than in the other two media. When the seven proteins were subjected to matrix-assisted laser desorption ionization-mass spectrometric analysis, four of the seven protein spots were unidentifiable. The other three proteins were identified as GroES heat shock protein, alpha antigen, and antigen 85 complex B (Ag85B; fibronectin-binding protein). These proteins may be associated with the heat resistance of M. paratuberculosis. Alpha antigen and Ag85B are both trehalose mycolyltransferases involved in mycobacterial cell wall assembly. TLC revealed that 7H9-OADC medium supported production of more trehalose dimycolates and cell wall-bound mycolic acids than did WR-GD medium. The present study shows that in vitro culture conditions significantly affect heat resistance, cell wall synthesis, and protein expression of M. paratuberculosis and emphasize the importance of culture conditions on in vitro and ex vivo studies to estimate heat resistance.     

Heterologous expression of Saccharomyces cerevisiae MPR1 gene confers tolerance to ethanol and l-azetidine-2-carboxylic acid in Hansenula polymorpha

Hansenula polymorpha is a naturally xylose-fermenting yeast; however, both its ethanol yield from xylose and ethanol resistance have to be improved before this organism can be used for industrial high-temperature simultaneous saccharification and fermentation of lignocellulosic materials. In the current research, we checked if the expression of the Saccharomyces cerevisiae MPR1 gene encoding N-acetyltransferase can increase the ethanol tolerance of H. polymorpha. The S. cerevisiae MPR1 gene was cloned in the H. polymorpha expression vector under the control of the H. polymorpha strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). H. polymorpha recombinant strains harboring 1–3 copies of the S. cerevisiae MPR1 gene showed enhanced tolerance to l-azetidine-2-carboxylic acid and ethanol. The obtained results suggest that the expression of the S. cerevisiae MPR1 gene in H. polymorpha can be a useful approach in the construction of H. polymorpha strains with improved ethanol resistance.     

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (¹³C, ¹⁵N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker’s yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine’s medium without lactic acid allows relatively low concentrations of ¹³C and ¹⁵N sources, and this medium can be reused several times with supplementation of the ¹³C source only.     

Hansenula polymorpha expressed heat shock protein gp96 exerts potent T cell activation activity as an adjuvant

Previous studies together with ours showed that heat shock protein gp96 as an adjuvant induces antigen specific T cell responses against cancer and infectious diseases. However, at present there is no efficient method to obtain high amount of full-length gp96 by in vitro expression. Here, we used the yeast Hansenula polymorpha as an efficient host for gp96 recombinant protein production. The transformant clones with highly expressed recombinant proteins were screened and selected by measuring the halo size which indicates enzymatic hydrolysis of starch in the medium. High-level production of gp96 (around 150 mg/mL) was achieved by using high-cell density fed-batch cultivations. We showed that peptide binding of the recombinant protein has similar specificity and intrinsic binding parameters as that of the native gp96. We next examined the self-assembly properties and high-order structures of the recombinant protein. Moreover, the H. polymorpha expressed recombinant gp96 can effectively induce HBV-specific CTL response in immunized mice while Escherichia coli-expressed gp96 cannot. Our results therefore may provide bases for structure and functional studies of gp96 and thereby potentially expedite the development of gp96-based vaccines for immunotherapy of cancer or infectious diseases.     

Induced release of membrane vesicles from rat adipocytes containing glycosylphosphatidylinositol-anchored microdomain and lipid droplet signalling proteins

Synthesis and degradation of lipids in mammalian adipocytes are tightly and coordinatedly regulated by insulin, fatty acids, reactive oxygen species and drugs. Conversely, the lipogenic or lipolytic state of adipocytes is communicated to other tissues by the secretion of soluble adipocytokines. Here we report that insulin, palmitate, H₂O₂ and the antidiabetic sulfonylurea drug glimepiride induce the release of the typical lipid droplet (LD) protein, perilipin-A, as well as typical plasma membrane microdomain (DIGs) proteins, such as caveolin-1 and the glycosylphosphatidylinositol (GPI)-anchored proteins, Gce1 and CD73 from rat adipocytes. According to biochemical and morphological criteria these LD and GPI-proteins are embedded within two different types of phospholipid-containing membrane vesicles, collectively called adiposomes. Adiposome release was not found to be causally related to cell lysis or apoptosis. The interaction of Gce1 and CD73 with the adiposomes apparently depends on their intact GPI anchor. Pull-down of caveolin-1, perilipin-A and CD73 together with phospholipids (via binding to annexin-V) as well as mutually of caveolin-1 with CD73 or perilipin-A (via coimmunoprecipitation) argues for their colocalization within the same adiposome vesicle.Taken together, certain lipogenic and anti-lipolytic agents induce the specific release of a subset of LD and DIGs proteins, including certain GPI-proteins, in adiposomes from primary rat adipocytes. Given the (c)AMP-degrading activities of Gce1 and CD73 and LD-forming function of perilipin-A and caveolin-1, the physiological relevance of the release of adiposomes from adipocytes may rely on the intercellular transfer of lipogenic and anti-lipolytic information.     

Metabolic engineering of the yeast Hansenula polymorpha for the construction of efficient ethanol producers

Until recently, the methylotrophic yeast has not been considered as a potential producer of biofuels, particularly, ethanol from lignocellulosic hydrolysates. The first work published 10 years ago revealed the ability of the thermotolerant methylotrophic yeast Hansenula polymorpha to ferment xylose—one of the main sugars of lignocellulosic hydrolysates—which has made the yeast a promising organism for high-temperature alcoholic fermentation. Such a feature of H. polymorpha could be used in the implementation of a potentially effective process of simultaneous saccharification and fermentation (SSF) of raw materials. SSF makes it possible to combine enzymatic hydrolysis of raw materials with the conversion of the sugars produced into ethanol: enzymes hydrolyze polysaccharides to monomers, which are immediately consumed by microorganisms (producers of ethanol). However, the efficiency of alcoholic fermentation of major sugars produced via hydrolysis of lignocellulosic raw materials and, especially, xylose by wild strains of H. polymorpha requires significant improvements. In this review, the main results of metabolic engineering of H. polymorpha for the construction of improved producers of ethanol from xylose, starch, xylan, and glycerol, as well as that of strains with increased tolerance to high temperatures and ethanol, are represented.     

A comparative study of the adjuvanticity of Hansenula polymorpha, Saccharomyces cerevsiae and Yarrowia lipolytica in oral and nasal immunization with virus capsid antigens

The adjuvanticity of Hansenula polymorpha, Saccharomyces cerevsiae and Yarrowia lipolytica were compared for oral and nasal immunization with virus capsid antigens. Mice were immunized orally with human papillomavirus type 16 L1 virus-like particles (HPV16 L1 VLPs), or intra-nasally with formalin-inactivated influenza A virus (FIV), in combination with one or other yeast. Mice receiving HPV16 L1 VLPs combined with H. polymorpha had a significantly higher titer for serum anti-HPV16 L1 IgG and neutralizing activity than those receiving HPV16 L1 VLPs combined with either of the other two yeasts. Also, mice receiving FIV combined with H. polymorpha had not only a markedly higher anti-influenza A virus IgG titer but also a higher survival rate after a potentially lethal influenza A virus challenge. We suggest that H. polymorpha thus will be useful for enhancing immune responses in mucosal immunizations.     

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.     

Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C

Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.