Killer toxin secretion through the cell wall of the yeastPichia anomala

A secreted killer toxin was detected through the cell wall ofPichia anomala cells by ultrastructural immunodetection with a specific monoclonal antibody (MAb KT4). MAb KT4 was successively detected by colloidal gold labeled streptavidin and biotinylated anti-mouse F(ab')₂ antibodies. The antigenic determinants of the toxin were localized throughout the cytoplasm and the cell wall of killer yeast cells. The Lowicryl K4M-immunogold method gave very satisfactory results and showed that the killer toxin was somewhat concentrated in the yeast cell wall layers before being exported into the medium. In agreement with previous reports, the binding of MAb KT4 suggested that theP. anomala killer toxin secretion did not result from a homogeneous diffusion across the yeast cell wall.Abbreviations G15 gold particles of 15 nm - IEM immunoelectron microscopy - IFA immunofluorescence assay - MAb monoclonal antibody - PBS phosphate buffered saline - SAM/F(ab)₂ sheep antibodies anti-mouse F(ab)₂ - SBB Sabouraud buffered broth     

Pichia anomala and Kluyveromyces wickerhamii killer toxins as new tools against Dekkera/Brettanomyces spoilage yeasts

Two yeast killer toxins active on spoilage yeasts belonging to the genus Dekkera/Brettanomyces are here described for the first time. The two toxins produced by Pichia anomala (DBVPG 3003) and Kluyveromyces wickerhamii (DBVPG 6077), and named Pikt and Kwkt, respectively, differ for molecular weight and biochemical properties. Interestingly, the fungicidal effect exerted by Pikt and Kwkt against Dekkera bruxellensis is stable for at least 10 days in wine. Thus, a potential application for the two toxins as antimicrobial agents active on Dekkera/Brettanomyces during wine ageing and storage can be hypothesised.     

Biotechnology, physiology and genetics of the yeast Pichia anomala

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.     

Characterization of the solution properties of Pichia farinosa killer toxin using PGSE NMR diffusion measurements

The solution behaviour with respect to pH and NaCl concentration of the tertiary structure and propensity for aggregation of salt- mediated killer toxin (SMKT) from Pichia farinosa was examined using pulsed-gradient spin-echo NMR diffusion measurements. It was found that in 0.15m NaCl the tertiary structure of SMKT was constant below pH 5.0, with the native SMKT existing as an unaggregated heterodimer containing the -subunit in a compactly folded form. However, above pH 5.0 the -subunit dissociated and lost its compact structure, becoming a random coil with an 37% increase in effective hydrodynamic radius. To determine the effects of NaCl concentration on the tertiary structure of SMKT, diffusion measurements were performed at pH 3.5 and NaCl concentrations up to 2M. Both the tertiary structure and aggregation state of SMKT were found to be insensitive to the salt concentration which indicates that the activity of the toxin is not a direct result of salt–protein interactions.     

New avenues for phage-display library to produce a Cryptococcus-specific anti-idiotypic antibody of HM-1 killer toxin

Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.     

Physiological characteristics of the biocontrol yeast Pichia anomala J121

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3 degrees C and 37 degrees C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.     

Genetic characterization of the yeast Pichia anomala (strain K), an antagonist of postharvest diseases of apple

AIMS: To obtain information about the genomic organization of Pichia anomala (strain K) and about its genomic diversity at species and intraspecies level. METHODS AND RESULTS: The PFGE karyotype of strain K was composed of four bands ranging in size from 1.1 to 3.2 Mb. The number of chromosomes was estimated at six since bands 2 and 3 seemed to result from the comigration of two chromosomes with similar size. A comparison of strain K and Hansenulawingeii migration profiles led to the estimate of K strain genome size at 11.7 Mb. Comparison with isogenic strains, resulting from the sporulation of strain K, highlighted some major karyotypic differences. Two segregants (KH6 and KH7) showed supernumerary chromosomes and one (KH9) displayed chromosomal length polymorphism. This genomic instability was confirmed by molecular hybridization with four probes, consisting of URA3, LEU2, PAEXG1 and PAEXG2 genes of P. anomala. URA3 and LEU2 probes showed second hybridization signals on supernumerary chromosomes of strain KH7 and on chromosome 6 of strain K for LEU2 only. Karyotypic comparison of seven non-isogenic P. anomala strains revealed chromosomal length polymorphism, a sign of intraspecies variation. CONCLUSIONS: This work has supplied information about genome size and chromosome number of strain K of P. anomala. The strain seems to be aneuploid because of the presence of supernumerary chromosomes and additional hybridization signals for URA3 and LEU2 probes in the chromosomal profile of some segregants. The work also highlighted genomic diversity within the P. anomala species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results obtained here increase information about the aneuploidy of P. anomala (strain K). Information about the genomic diversity of the segregants will be of great interest for further studies on strain K mode of action. The genome size and chromosomal profile of P. anomala presented here are different from the results obtained elsewhere for Hansenula anomala, while Hansenula is included as a synonym of Pichia. This warrants further studies to investigate this taxonomic relationship.     

Oxoester oxidoreductase activities in new isolates of Pichia anomala from apple, grape and cane juices

Thirty-nine yeasts isolated from apple, grape and cane juices were screened for their oxidoreductase activity. The two strains of Pichia, one isolated from apple and one from cane juices, appear to be promising strains for oxidoreductase activity on alpha-oxoesters. They showed similar high yields in converting ethyl pyruvate to ethyl lactate as Saccharomyces spp. (86.6% and 85.3% versus 86.6%), and higher yields in the reduction of alpha-oxocarboxylic esters (ketopantolactone to pantolactone: 74% and 73.3%, respectively) compared to Saccharomyces spp. (yield 60%).     

Improvement in cell‐bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell‐bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X‐100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X‐100 for 30 min resulted in c. 15% enhancement in cell‐bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X‐100 led to enhancement in cell‐bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.     

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 °C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.     

Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

AIMS: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. METHODS AND RESULTS: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from -20 to +30 degrees C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10 degrees C, 75-90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (-20 to 30 degrees C). Trehalose was the most effective protectant at 20 and 30 degrees C (>20% viable cells after 12 weeks at 20 degrees C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. CONCLUSIONS: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.     

High-level secretory expression of immunologically active intact antibody from the yeast Pichia pastoris

We have produced a functional murine antibody to dioxin in the culture medium of the methylotrophic yeast Pichia pastoris. Complementary DNA copies encoding the light () and heavy () chains of the dioxin monoclonal antibody, DD1, were each placed under the control of P.pastoris alcohol oxidase (AOX1) promoter and Saccharomyces cerevisiae -mating factor secretion signal sequence. The resulting expression cassettes were assembled into a single plasmid (pPICZDD1) to permit co-expression of both light and heavy chains of the antibody molecule. P.pastoris SMD1168 (pep4, his4) transformed with pPICZDD1 was able to secrete intact antibody into the culture medium. As high as 36 mg l^–1 of the antibody was produced in shake-flask cultures after 96-h induction with methanol. Functional analysis using immunoassay confirmed murine nature of the recombinant antibody and its ability to bind dioxin.     

In vitro susceptibilities of Candida spp. to Panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434

Panomycocin, the killer toxin of Pichia anomala NCYC 434 (K5), is a 49 kDa monomeric glycoprotein with exo-beta-1,3-glucanase activity (patent pending). In this study we evaluated the in vitro activity of panomycocin against a panel of 109 human isolates of seven different pathogenic Candida spp. using microdilution and time-kill methods. Panomycocin was most active against C. tropicalis, C. pseudotropicalis and C. glabrata with MIC(90) values of 1 microg/ml. It displayed significant activity against C. albicans and C. parapsilosis with MIC(90) values of 4 and 2 microg/ml, respectively. For C. krusei, the MIC(90) value was 8 microg/ml. Panomycocin was fungicidal against all the tested Candida spp. The MFC values were only one or 2 dilutions higher than the MICs with the exception of C. krusei isolates with MFCs greater than or equal to 4xMIC. Results of this study indicated that panomycocin could be considered as a natural antifungal agent against Candida infections and has significant potential for further investigation.     

甲醇-山梨醇混合碳源诱导提高抗HER2抗体在糖基工程毕赤酵母中的表达

目的：以抗HER2抗体为模型,研究抗体在糖基工程酵母菌中的表达及工程菌发酵技术。方法：首先通过摇瓶试验分析诱导用甲醇浓度对抗体表达的影响,并用高表达HER2的SK-BR-3细胞分析抗HER2抗体的抗原结合活性。以此为基础,在5 L发酵罐中研究甲醇-山梨醇混合碳源流加诱导对抗HER2抗体表达水平的影响;收集发酵培养液,采用阳离子交换层析对目标产物进行纯化;利用SDS-PAGE、Western印迹、Lowry法对抗体的相对分子质量、浓度等进行分析。结果：摇瓶试验结果表明,甲醇浓度为0.5%时抗体表达量最高,且糖基工程毕赤酵母菌表达的抗HER2抗体具有与SK-BR-3细胞抗原结合的活性;在5 L发酵罐中,利用甲醇和山梨醇混合诱导方式发酵表达抗体,其表达量可提高至0.6 g/L,比摇瓶诱导表达的抗体产量提高了近10倍;非还原SDS-PAGE及Western印迹表明抗体相对分子质量为1.5×105,与商业化抗体Herceptin的大小一致;经过一步阳离子交换层析纯化,纯化后抗体浓度为0.365 g/L。结论：采用甲醇-山梨醇混合碳源诱导方式在5 L发酵罐中进行发酵表达,能够提高抗HER2抗体在糖基工程酵母菌中的表达量,本研究可为抗体在酵母中的规模发酵技术提供重要参考。     

Purification and characterization of a thermostable and acid-stable phytase from Pichia anomala

Phytase of Pichia anomala was purified to near homogeneity by a two-step process of acetone precipitation followed by anion exchange chromatography using DEAE-Sephadex. The enzyme had a molecular weight of 64 kDa. It was optimally active at 60 °C and pH 4.0. This enzyme was found to be highly thermostable and acid-stable, with a half life of 7 and 8 days at 60 °C and pH 4.0 respectively. At 80 °C, the half life of phytase could be increased from 5 to 30 min by the addition of materials such as sucrose, lactose and arabinose (10% w/v). The enzyme exhibited a broad substrate specificity, since it acted on p-nitrophenyl phosphate, ATP, ADP, glucose-6-phosphate besides phytic acid. The K _m value for phytic acid was 0.20 mM and V _max was 6.34 mol/mg protein/min. There was no requirement of metal ions for activity. SDS was observed to be highly inhibitory to phytase activity. Sodium azide, DTT, -mercaptoethanol, EDTA, toluene, glycerol, PMSF, iodo-acetate and N-bromosuccinimide did not show inhibitory activity. The enzyme was inhibited by 2,3-butanedione, indicating the involvement of arginine residues in catalysis. Phytase activity was not inhibited in the presence of inorganic phosphate upto 10 mM. The shelf life of the enzyme was 6 months at 4 °C and there was no loss in the activity on lyophilization. Very few studies have been done on purification of yeast phytases. This is the first report on purification and characterization of phytase from P. anomala. The enzyme is unique in being thermostable, acid-stable, exhibiting broad substrate specificity and in not requiring metal ions for its activity. The yeast biomass containing phytase appears to be suitable for supplementing animal feeds to improve the availability of phosphorus from phytates.     

The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531

In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 × CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of⁵¹Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 × CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.     

Serological study of yeast killer toxins by monoclonal antibodies

Yeast killer toxins coded by determined and undetermined killer plasmids or presumptive nuclear gene(s) in various genera (Saccharomyces, Kluyveromyces, Pichia and Candida) have been serologically investigated by a monoclonal antibody (KT4), produced against the yeast killer toxin of Pichia (Hansenula) anomala UCSC 25F. Double immunodiffusion with the killer toxins as antigens and indirect immunofluorescence on whole cells of the corresponding killer yeast have been used. In both the serological procedures, monoclonal antibody KT4 proved to be reacting only with the killer toxins and the whole cells of yeasts belonging to the genus Pichia.