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Andriy Sibirny - One of the best experts on this subject based on the ideXlab platform.
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Development of new dominant selectable markers for the nonconventional yeasts Ogataea polymorpha and Candida famata.
Yeast (Chichester England), 2020Co-Authors: Dmytro Bratiichuk, Olena O. Kurylenko, Kostyantyn V. Dmytruk, Roksolana Vasylyshyn, Mingxing Zuo, Yingqian Kang, Andriy SibirnyAbstract:Nonconventional yeast Candida famata and Ogataea polymorpha are interesting organisms for basic and applied studies. O. polymorpha is methylotrophic thermotolerant yeast capable of xylose alcoholic fermentation whereas C. famata is capable of riboflavin overproduction. Still, the new tools for molecular research of these species are needed. The aim of this study was to develop the new dominant selective markers for C. famata and O. polymorpha usable in metabolic engineering experiments. In this work, the BSD gene from Aspergillus terreus coding for blasticidin S deaminase, O. polymorpha AUR1 gene required for sphingolipid synthesis and IMH3 gene, which encodes IMP dehydrogenase, were tested as the new dominant selective marker genes. Our results showed that AUR1 and IMH3 genes could be used as dominant selective markers for O. polymorpha with frequencies of transformation of 40 and 20 transformants per microgram of DNA, respectively. The IMH3 gene was successfully used as the marker for construction of O. polymorpha strains with increased ethanol production from xylose due to overexpression of TAL1, TKL1 and AOX1 genes. The BSD gene from A. terreus, conferring resistance to blasticidin, was found to be efficient for selection of C. famata transformants.
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Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha)
Journal of Industrial Microbiology & Biotechnology, 2020Co-Authors: Justyna Ruchala, Kostyantyn V. Dmytruk, Olena O. Kurylenko, Andriy SibirnyAbstract:This review summarizes progress in the construction of efficient yeast ethanol producers from glucose/sucrose and lignocellulose. Saccharomyces cerevisiae is the major industrial producer of first-generation ethanol. The different approaches to increase ethanol yield and productivity from glucose in S. cerevisiae are described. Construction of the producers of second-generation ethanol is described for S. cerevisiae , one of the best natural xylose fermenters, Scheffersomyces stipitis and the most thermotolerant yeast known Ogataea polymorpha . Each of these organisms has some advantages and drawbacks. S. cerevisiae is the primary industrial ethanol producer and is the most ethanol tolerant natural yeast known and, however, cannot metabolize xylose. S. stipitis can effectively ferment both glucose and xylose and, however, has low ethanol tolerance and requires oxygen for growth. O. polymorpha grows and ferments at high temperatures and, however, produces very low amounts of ethanol from xylose. Review describes how the mentioned drawbacks could be overcome.
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Anhydrobiosis in yeasts: Glutathione synthesis by yeast Ogataea (Hansenula) polymorpha cells after their dehydration-rehydration.
Journal of biotechnology, 2019Co-Authors: Olena O. Kurylenko, Andriy Sibirny, Justyna Ruchala, Linda Rozenfelde, Galina Khroustalyova, Roksolana Vasylyshyn, Chuang-rung Chang, Rimantas Daugelavičius, Alexander RapoportAbstract:The possibility of using active dry microbial preparations in biotechnological processes is essential for the development of new modern industrial technologies. In this study, we show the possibility of obtaining such preparations of the genetically engineered yeast strain Ogataea (Hansenula) polymorpha with glutathione overproduction. Special pre-treatment involving the gradual rehydration of dry cells in water vapour led to the restoration/reactivation of almost 100% of dehydrated cells. Furthermore, dry cells do not lose their viability during storage at room temperatures. Application of dry cells as the inoculum provides the same levels of glutathione synthesis as that of a native yeast culture.
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Overexpression of the genes of glycerol catabolism and glycerol facilitator improves glycerol conversion to ethanol in the methylotrophic thermotolerant yeast Ogataea polymorpha.
Yeast (Chichester England), 2019Co-Authors: Marta Semkiv, Kostyantyn V. Dmytruk, Iwona Kata, Orysya Ternavska, Wladimir Sibirny, Andriy SibirnyAbstract:Production of fuel ethanol is one of the possible ways to utilize crude glycerol, substantial amounts of which are produced by biodiesel industry. Earlier, we have described construction of the recombinant strains of methylotrophic thermotolerant yeast Ogataea polymorpha with simultaneous overexpression of the genes PDC1 and ADH1, which produced increased amounts of ethanol from glycerol. In this work, we have further improved these strains by overexpression of genes involved either in oxidative (through dihydroxyacetone) or phosphorylative (through glycerol-3-phosphate) pathway of glycerol catabolism, as well as heterologous gene coding for glycerol transporter FPS1 from Komagataella phaffii (formerly, Pichia pastoris). Obtained recombinant strains produced up to 10.7 g/L of ethanol (with ethanol productivity 30 mg/g of biomass/hr and yield 132 mg/g of consumed glycerol) from pure glycerol and up to 3.55 g/L of ethanol (with ethanol productivity 11.6 mg/g of biomass/hr and yield 72.3 mg/g of consumed glycerol) from crude glycerol as a carbon source, which is approximately 15 times more relative to that of the O. polymorpha wild-type strain and 2.2 more relative to the earlier constructed strain.
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Glutathione Metabolism in Yeasts and Construction of the Advanced Producers of This Tripeptide
Non-conventional Yeasts: from Basic Research to Application, 2019Co-Authors: Olena O. Kurylenko, Kostyantyn V. Dmytruk, Andriy SibirnyAbstract:Glutathione is the most abundant non-protein thiol compound of the most living organisms able to protect cells from nutritional, environmental, and oxidative stresses. Due to the antioxidative properties, glutathione is widely used as an active ingredient of drugs, food, and cosmetic products. Microbial synthesis using yeasts is currently the most common method for the commercial production of glutathione. Construction of glutathione overproducers in yeasts by metabolic engineering approaches and optimization of the technology for its production has potential to satisfy the increasing industrial demand in this tripeptide. This review summarizes the current knowledge of physiological functions and practical applications of glutathione as well as illustrates strategies for its efficient production. The potential of the methylotrophic yeast Ogataea polymorpha as a glutathione producer is also discussed.
Niran Roongsawang - One of the best experts on this subject based on the ideXlab platform.
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Protein secretion in wild-type and Othac1 mutant strains of thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.
Molecular biology reports, 2019Co-Authors: Chitwadee Phithakrotchanakoon, Sutipa Tanapongpipat, Narumon Phaonakrop, Sittiruk Roytrakul, Niran RoongsawangAbstract:In yeasts, Hac1 transcription factor of the unfolded protein response (UPR) regulates many genes involved in secretory pathways. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a host for heterologous protein secretion. To understand the role of OtHac1 on the secretome of O. thermomethanolica, a comparative proteomic analysis using LC–MS/MS was employed to identify proteins with altered secretion levels when OtHac1 was mutated. 268 proteins were detected in the extracellular medium of O. thermomethanolica wild-type control and Othac1 mutant strains. A number of metabolic enzymes functioning in amino acid, carbohydrate, glycan, and lipid metabolism showed altered secretion in the mutant suggesting that OtHac1 may play a role in mediating extracellular metabolism. Most of the extracellular proteins identified do not contain canonical signal sequences suggesting that they are secreted via unconventional protein secretion pathways. Collectively, the data provide insights into protein secretion and OtHac1 function in O. thermomethanolica which will be useful for developing efficient host for protein production.
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Sucrose-inducible heterologous expression of phytase in high cell density cultivation of the thermotolerant methylotrophic yeast Ogataea thermomethanolica.
FEMS microbiology letters, 2019Co-Authors: Kriengsak Boonchoo, Sutipa Tanapongpipat, Aekkachai Puseenam, Kanokarn Kocharin, Niran RoongsawangAbstract:In this study, production of fungal phytase in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 employing methanol-inducible OtAOX promoter and sucrose-inducible OtMal promoter was investigated in a high cell density fed-batch fermentation. Although a similar maximum cell concentration was obtained in both expression systems, the OtMal system gave ~2-fold higher phytase activity, specific yield, production yield, volumetric productivity and specific productivity rate compared with the OtAOX system. In addition to being more efficient, the OtMal system is more flexible because sucrose or sugarcane molasses can be utilized as less expensive carbon sources instead of glycerol in batch and fed-batch stages. Phytase yields from the OtMal system produced using sucrose or sugarcane molasses are comparable with those obtained with glycerol. We estimate the cost of phytase production by the OtMal system using sucrose or sugarcane molasses to be ~85% lower than the OtAOX system.
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A novel sucrose-based expression system for heterologous proteins expression in thermotolerant methylotrophic yeast Ogataea thermomethanolica.
FEMS microbiology letters, 2018Co-Authors: Aekkachai Puseenam, Lily Eurwilaichitr, Supawadee Ingsriswang, Sutipa Tanapongpipat, Kanokarn Kocharin, Niran RoongsawangAbstract:The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein expression. In this study, a novel expression system was developed for O. thermomethanolica based on the maltase (mal) gene promoter from this organism. The OtMal promoter function was tested for expression of fungal enzymes as reporter genes. Measurement of xylanase reporter enzyme activity showed that the OtMal promoter was repressed during growth on glucose and was activated by sucrose. When sucrose was used as a carbon source, the OtMal promoter was approximately twice as strong as the constitutive OtGAP promoter. Comparison of the OtMal promoter with the methanol-inducible OtAOX promoter showed that OtMal promoter drove 1.2 and 1.7-fold higher expression of xylanase and phytase reporter, respectively, than OtAOX promoter under inducing conditions at 24 h. Our results indicated that this novel expression system could be useful for the production of heterologous proteins from sucrose in yeast O. thermomethanolica.
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Synthesis and characterization of Ogataea thermomethanolica alcohol oxidase immobilized on barium ferrite magnetic microparticles.
Journal of bioscience and bioengineering, 2018Co-Authors: Natthaya Mangkorn, Pattanop Kanokratana, Niran Roongsawang, Navadol Laosiripojana, Apirat Laobuthee, Verawat ChampredaAbstract:Alcohol oxidase catalyzes the oxidation of primary alcohols into the corresponding aldehydes, making it a potential biocatalyst in the chemical industry. However, the high production cost and poor operational stability of this enzyme are limitations for industrial application. Immobilization of enzyme onto solid supports is a useful strategy for improving enzyme stability. In this work, alcohol oxidase from the thermotolerant methylotrophic yeast Ogataea thermomethanolica (OthAOX) was covalently immobilized onto barium ferrite (BaFe12O19) magnetic microparticles. Among different conditions tested, the highest immobilization efficiency of 71.0 % and catalytic activity of 34.6 U/g was obtained. Immobilization of OthAOX onto magnetic support was shown by Fourier-Transformed infrared microscopy, scanning electron microscopy and X-ray diffraction. The immobilized OthAOX worked optimally at 55 °C and pH 8.0. Immobilization also improved thermostability, in which >65% of the initial immobilized enzyme activity was retained after 24 h pre-incubation at 45 °C. The immobilized enzyme showed a greater catalytic efficiency for oxidation of methanol and ethanol than free enzyme. The immobilized enzyme could be recovered by magnetization and recycled for at least three consecutive batches, after which 70% activity remained. The properties of the immobilized enzyme suggest its potential industrial application for synthesis of aldehyde.
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Hac1 function revealed by the protein expression profile of a OtHAC1 mutant of thermotolerant methylotrophic yeast Ogataea thermomethanolica.
Molecular biology reports, 2018Co-Authors: Chitwadee Phithakrotchanakoon, Sutipa Tanapongpipat, Aekkachai Puseenam, Narumon Phaonakrop, Sittiruk Roytrakul, Niran RoongsawangAbstract:In yeast, the accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR) pathway, which is mediated by Hac1 transcription factor. Here, we characterized the function of a gene encoding Hac1 in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 (OtHAC1). OtHAC1 mRNA contains a non-canonical intron of 176 nt, which was demonstrated to be spliced by RT-PCR. To characterize the function of this gene, we compared the proteome of a Othac1 mutant with wild-type. A total of 463 proteins with differential abundance were detected. The functions of these proteins were annotated in oxidative stress, metabolic pathways, transcription, translation, and of particular interest in secretory pathway. While many intracellular proteins differentially expressed in the mutant were similar to proteins with altered expression in UPR-stressed Saccharomyces cerevisiae, two novel OtHAC1-dependent proteins (Iml1 and Npr2) were identified that are potentially involved in the regulation of autophagy. The data show that OtHAC1 is an important regulator of several different processes in O. thermomethanolica TBRC656.
Sutipa Tanapongpipat - One of the best experts on this subject based on the ideXlab platform.
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Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656.
Microbiological research, 2019Co-Authors: Sriwan Wongwisansri, Yoshinobu Kaneko, Peerada Promdonkoy, Somsak Likhitrattanapisal, Piyanun Harnpichanchai, Kazuhito Fujiyama, Lily Eurwilaichitr, Supawadee Ingsriswang, Sutipa TanapongpipatAbstract:The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.
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Protein secretion in wild-type and Othac1 mutant strains of thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656.
Molecular biology reports, 2019Co-Authors: Chitwadee Phithakrotchanakoon, Sutipa Tanapongpipat, Narumon Phaonakrop, Sittiruk Roytrakul, Niran RoongsawangAbstract:In yeasts, Hac1 transcription factor of the unfolded protein response (UPR) regulates many genes involved in secretory pathways. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a host for heterologous protein secretion. To understand the role of OtHac1 on the secretome of O. thermomethanolica, a comparative proteomic analysis using LC–MS/MS was employed to identify proteins with altered secretion levels when OtHac1 was mutated. 268 proteins were detected in the extracellular medium of O. thermomethanolica wild-type control and Othac1 mutant strains. A number of metabolic enzymes functioning in amino acid, carbohydrate, glycan, and lipid metabolism showed altered secretion in the mutant suggesting that OtHac1 may play a role in mediating extracellular metabolism. Most of the extracellular proteins identified do not contain canonical signal sequences suggesting that they are secreted via unconventional protein secretion pathways. Collectively, the data provide insights into protein secretion and OtHac1 function in O. thermomethanolica which will be useful for developing efficient host for protein production.
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Sucrose-inducible heterologous expression of phytase in high cell density cultivation of the thermotolerant methylotrophic yeast Ogataea thermomethanolica.
FEMS microbiology letters, 2019Co-Authors: Kriengsak Boonchoo, Sutipa Tanapongpipat, Aekkachai Puseenam, Kanokarn Kocharin, Niran RoongsawangAbstract:In this study, production of fungal phytase in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 employing methanol-inducible OtAOX promoter and sucrose-inducible OtMal promoter was investigated in a high cell density fed-batch fermentation. Although a similar maximum cell concentration was obtained in both expression systems, the OtMal system gave ~2-fold higher phytase activity, specific yield, production yield, volumetric productivity and specific productivity rate compared with the OtAOX system. In addition to being more efficient, the OtMal system is more flexible because sucrose or sugarcane molasses can be utilized as less expensive carbon sources instead of glycerol in batch and fed-batch stages. Phytase yields from the OtMal system produced using sucrose or sugarcane molasses are comparable with those obtained with glycerol. We estimate the cost of phytase production by the OtMal system using sucrose or sugarcane molasses to be ~85% lower than the OtAOX system.
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A novel sucrose-based expression system for heterologous proteins expression in thermotolerant methylotrophic yeast Ogataea thermomethanolica.
FEMS microbiology letters, 2018Co-Authors: Aekkachai Puseenam, Lily Eurwilaichitr, Supawadee Ingsriswang, Sutipa Tanapongpipat, Kanokarn Kocharin, Niran RoongsawangAbstract:The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 is a potential host for heterologous protein expression. In this study, a novel expression system was developed for O. thermomethanolica based on the maltase (mal) gene promoter from this organism. The OtMal promoter function was tested for expression of fungal enzymes as reporter genes. Measurement of xylanase reporter enzyme activity showed that the OtMal promoter was repressed during growth on glucose and was activated by sucrose. When sucrose was used as a carbon source, the OtMal promoter was approximately twice as strong as the constitutive OtGAP promoter. Comparison of the OtMal promoter with the methanol-inducible OtAOX promoter showed that OtMal promoter drove 1.2 and 1.7-fold higher expression of xylanase and phytase reporter, respectively, than OtAOX promoter under inducing conditions at 24 h. Our results indicated that this novel expression system could be useful for the production of heterologous proteins from sucrose in yeast O. thermomethanolica.
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Hac1 function revealed by the protein expression profile of a OtHAC1 mutant of thermotolerant methylotrophic yeast Ogataea thermomethanolica.
Molecular biology reports, 2018Co-Authors: Chitwadee Phithakrotchanakoon, Sutipa Tanapongpipat, Aekkachai Puseenam, Narumon Phaonakrop, Sittiruk Roytrakul, Niran RoongsawangAbstract:In yeast, the accumulation of unfolded proteins in the ER triggers the unfolded protein response (UPR) pathway, which is mediated by Hac1 transcription factor. Here, we characterized the function of a gene encoding Hac1 in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 (OtHAC1). OtHAC1 mRNA contains a non-canonical intron of 176 nt, which was demonstrated to be spliced by RT-PCR. To characterize the function of this gene, we compared the proteome of a Othac1 mutant with wild-type. A total of 463 proteins with differential abundance were detected. The functions of these proteins were annotated in oxidative stress, metabolic pathways, transcription, translation, and of particular interest in secretory pathway. While many intracellular proteins differentially expressed in the mutant were similar to proteins with altered expression in UPR-stressed Saccharomyces cerevisiae, two novel OtHAC1-dependent proteins (Iml1 and Npr2) were identified that are potentially involved in the regulation of autophagy. The data show that OtHAC1 is an important regulator of several different processes in O. thermomethanolica TBRC656.
Yoshinobu Kaneko - One of the best experts on this subject based on the ideXlab platform.
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Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656.
Microbiological research, 2019Co-Authors: Sriwan Wongwisansri, Yoshinobu Kaneko, Peerada Promdonkoy, Somsak Likhitrattanapisal, Piyanun Harnpichanchai, Kazuhito Fujiyama, Lily Eurwilaichitr, Supawadee Ingsriswang, Sutipa TanapongpipatAbstract:The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.
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efficient genome editing by crispr cas9 with a trna sgrna fusion in the methylotrophic yeast Ogataea polymorpha
Journal of Bioscience and Bioengineering, 2017Co-Authors: Minori Numamoto, Hiromi Maekawa, Yoshinobu KanekoAbstract:The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.
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Polo-like kinase Cdc5 regulates Spc72 recruitment to spindle pole body in the methylotrophic yeast Ogataea polymorpha.
eLife, 2017Co-Authors: Hiromi Maekawa, Annett Neuner, Diana Rüthnick, Elmar Schiebel, Gislene Pereira, Yoshinobu KanekoAbstract:Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha.
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Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha.
Journal of bioscience and bioengineering, 2017Co-Authors: Minori Numamoto, Hiromi Maekawa, Yoshinobu KanekoAbstract:The methylotrophic yeast Ogataea polymorpha (syn. Hansenula polymorpha) is an attractive industrial non-conventional yeast showing high thermo-tolerance (up to 50°C) and xylose assimilation. However, genetic manipulation of O. polymorpha is often laborious and time-consuming because it has lower homologous recombination efficiency relative to Saccharomyces cerevisiae. To overcome this disadvantage, we applied the CRISPR/Cas9 system as a powerful genome editing tool in O. polymorpha. In this system, both single guide RNA (sgRNA) and endonuclease Cas9 were expressed by a single autonomously-replicable plasmid and the sgRNA portion could be easily changed by using PCR and In-Fusion cloning techniques. Because the mutation efficiency of the CRISPR/Cas9 system was relatively low when the sgRNA was expressed under the control of the OpSNR6 promoter, the tRNACUG gene was used for sgRNA expression. The editing efficiency of this system ranged from 17% to 71% of transformants in several target genes tested (ADE12, PHO1, PHO11, and PHO84). These findings indicate that genetic manipulation of O. polymorpha will be more convenient and accelerated by using this CRISPR/Cas9 system.
Yasunori Chiba - One of the best experts on this subject based on the ideXlab platform.
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Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta.
Journal of bioscience and bioengineering, 2018Co-Authors: Takehiko Yoko-o, Akiko Komatsuzaki, Erina Yoshihara, Mariko Umemura, Yasunori ChibaAbstract:Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta also possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of therapeutic glycoproteins. However, genetic methods, which are required for the construction of strains by breeding, have not yet been established in this organism. In this study, we investigated the O. minuta mechanisms of mating and sporulation, which would facilitate genetic analysis in this species. Specifically, we determined DNA sequences around the MAT locus in O. minuta strain NBRC 10746, and found that two MAT loci were flanked by a pair of inverted repeat sequences, as reported in O.polymorpha (Maekawa and Kaneko, PLOS Genet., 10, e1004796, 2014). As in O. polymorpha, mating type in O. minuta appears to be switched by inversion of the chromosomal region between the two MAT loci. We successfully obtained O. minuta diploid cells, which showed vegetative growth on rich medium. The size of the diploid cells was 1.3-fold larger than haploid cells of this species. Diploid cells formed ascospores, which contained 2-4 spores, under nutrient starvation conditions. Phenotypes of the resultant haploid cells exhibited Mendelian segregation, indicating that genetic approaches are applicable to O. minuta.
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Structural modeling and mutagenesis of endo-β-N-acetylglucosaminidase from Ogataea minuta identifies the importance of Trp295 for hydrolytic activity.
Journal of bioscience and bioengineering, 2017Co-Authors: Toshihiko Kitajima, Akiko Komatsuzaki, Yuanling Jia, Juan Cui, Fumiko Matsuzawa, Seiichi Aikawa, Xiao-dong Gao, Yasunori ChibaAbstract:Endo-β-N-acetylglucosaminidase from the methylotrophic yeast Ogataea minuta (Endo-Om) is a glycoside hydrolase family 85 enzyme that has dual catalytic activity in the hydrolysis and transglycosylation of complex N-glycans, in common with the enzymes from the eukaryotic species. In this study, we have conducted mutagenesis of Endo-Om at Trp295, to determine the effect on hydrolytic activity. Structural modeling predicted that Trp295 forms an important interaction with the α-1,3-linked mannose residue of the trimannosyl N-glycan core, rather than being directly involved in catalytic activity. Our results showed that an aromatic amino acid is required at position 295 for the hydrolytic activity of this enzyme. Notably, the tryptophan residue is highly conserved in eukaryotic endo-β-N-acetylglucosaminidases that show activity toward complex oligosaccharides. Accordingly, our results strongly suggested that Trp295 is involved in the recognition of oligosaccharide substrates by Endo-Om.
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Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones
Journal of Bioscience and Bioengineering, 2017Co-Authors: Takeshi Suzuki, Kimihisa Ichikawa, Rie Ito, Minako Ono, Masayuki Yabuta, Satoshi Baba, Koichi Nonaka, Yasunori ChibaAbstract:A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta.
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Identification and characterization of endo-β-N-acetylglucosaminidase from methylotrophic yeast Ogataea minuta
Glycobiology, 2013Co-Authors: Satoshi Murakami, Yuki Takaoka, Hisashi Ashida, Kenji Yamamoto, Hisashi Narimatsu, Yasunori ChibaAbstract:In four yeast strains, Ogataea minuta, Candida parapolymorpha, Pichia anomala and Zygosaccharomyces rouxii, we identified endo-β-N-acetylglucosaminidase (ENGase) homologous sequences by database searches; in each of the four species, a corresponding enzyme activity was also confirmed in crude cell extract obtained from each strain. The O. minuta ENGase (Endo-Om)-encoding gene was directly amplified from O. minuta genomic DNA and sequenced. The Endo-Om-encoding gene contained a 2319-bp open-reading frame; the deduced amino acid sequence indicated that the putative protein belonged to glycoside hydrolase family 85. The gene was introduced into O. minuta, and the recombinant Endo-Om was overexpressed and purified. When the enzyme assay was performed using an agalacto-biantennary oligosaccharide as a substrate, Endo-Om exhibited both hydrolysis and transglycosylation activities. Endo-Om exhibited hydrolytic activity for high-mannose, hybrid, biantennary and (2,6)-branched triantennary N-linked oligosaccharides, but not for tetraantennary, (2,4)-branched triantennary, bisecting N-acetylglucosamine structure and core-fucosylated biantennary N-linked oligosaccharides. Endo-Om also was able to hydrolyze N-glycans attached to RNase B and human transferrin under both denaturing and nondenaturing conditions. Thus, the present study reports the detection and characterization of a novel yeast ENGase.
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antibody expression in protease deficient strains of the methylotrophic yeast Ogataea minuta
Fems Yeast Research, 2007Co-Authors: Kousuke Kuroda, Toshihiro Komeda, Kazuhiro Motoki, Haruhiko Tsumura, Yasunori Chiba, Yoshinori Kitagawa, Eiji Mori, Kazuo Kobayashi, Shiro Kataoka, Yoshifumi JigamiAbstract:When human antibody genes were expressed in the methylotrophic yeast Ogataea minuta, the secreted antibody became partially degraded. To suppress the degradation, a vacuolar protease-deficient strain was constructed and its antibody production was evaluated. Although antibody productivity was improved in the vacuolar protease-deficient strain, the secreted antibody still became partially degraded. Peptide sequencing revealed that the cleavage occurred in the CH1 region of the heavy chain, implying that the cleavage was caused by an aspartic protease, Yps1p. To inhibit this cleavage, Yps1p-deficient strains were constructed and their antibody production was evaluated. As a result, the partial degradation of the antibody was suppressed in the O. minuta multiple-protease-deficient strains.
