[en] Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted.
Disciplines :
Biotechnology
Author, co-author :
Berrios, Julio ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso 2340000, Chile
Theron, Chrispian ; Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Microbial technologies ; GeneMill, Institute of Systems, Molecular and Integrative Biology, Biosciences Building, University of Liverpool, Crown Street, Liverpool L69 7BE, UK
Steels, Sébastien ; Université de Liège - ULiège > Département GxABT > Microbial technologies
Ponce, Belén ; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso 2340000, Chile
Velastegui, Edgar; School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso 2340000, Chile ; Microbial Processes and Interactions, TERRA Teaching and Research Centre, Gembloux Agro-Bio Tech, University of Liège, Av. de la Faculté 2B, 5030 Gembloux, Belgium
Bustos C. Quezada J. Veas R. Altamirano C. Braun-Galleani S. Fickers P. Berrios J. Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production Metabolites 2022 12 346 10.3390/metabo12040346 35448535
Gomes A.M.V. Carmo T.S. Carvalho L.S. Bahia F.M. Parachin N.S. Comparison of Yeasts as Hosts for Recombinant Protein Production Microorganisms 2018 6 38 10.3390/microorganisms6020038
Ohsawa S. Oku M. Yurimoto H. Sakai Y. Regulation of Peroxisome Homeostasis by Post-Translational Modification in the Methylotrophic Yeast Komagataella phaffii Front. Cell Dev. Biol. 2022 10 887806 10.3389/fcell.2022.887806 35517506
North M. Gaytán B.D. Romero C. Jr. de la Rosa V.Y. Loguinov A. Smith M.T. Zhang L. Vulpe C.D. Functional Toxicogenomic Profiling Expands Insight into Modulators of Formaldehyde Toxicity in Yeast Front. Genet. 2016 7 200 10.3389/fgene.2016.00200
Vanz A. Lünsdorf H. Adnan A. Nimtz M. Gurramkonda C. Khanna N. Rinas U. Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: Catabolic adaptation, stress responses, and autophagic processes Microb. Cell Factories 2012 11 103 10.1186/1475-2859-11-103 22873405
Jungo C. Marison I. von Stockar U. Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures J. Biotechnol. 2007 130 236 246 10.1016/j.jbiotec.2007.04.004 17566583
Wakayama K. Yamaguchi S. Takeuchi A. Mizumura T. Ozawa S. Tomizuka N. Hayakawa T. Nakagawa T. Regulation of intracellular formaldehyde toxicity during methanol metabolism of the methylotrophic yeast Pichia methanolica J. Biosci. Bioeng. 2016 122 545 549 10.1016/j.jbiosc.2016.03.022
Rußmayer H. Buchetics M. Gruber C. Valli M. Grillitsch K. Modarres G. Guerrasio R. Klavins K. Neubauer S. Drexler H. et al. Systems-level organization of yeast methylotrophic lifestyle BMC Biol. 2015 13 80 10.1186/s12915-015-0186-5
Lee B. Yurimoto H. Sakai Y. Kato N. Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the methylotrophic yeast Candida boidinii Microbiology 2002 148 2697 2704 12213916 10.1099/00221287-148-9-2697
Yano T. Takigami E. Yurimoto H. Sakai Y. Yap1-Regulated Glutathione Redox System Curtails Accumulation of Formaldehyde and Reactive Oxygen Species in Methanol Metabolism of Pichia pastoris Eukaryot Cell 2009 8 540 549 19252120 10.1128/EC.00007-09
Fickers P. le Dall M.T. Gaillardin C. Thonart P. Nicaud J.M. New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica J. Microbiol. Methods 2003 55 727 737 10.1016/j.mimet.2003.07.003 14607415
Lin-Cereghino J. Wong W.W. Xiong S. Giang W. Luong L.T. Vu J. Johnson S.D. Lin-Cereghino G.P. Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris Biotechniques 2005 38 44 48 10.2144/05381BM04 15679083
Theron C.W. Berrios J. Steels S. Telek S. Lecler R. Rodriguez C. Fickers P. Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris Yeast 2019 36 285 296 10.1002/yea.3388 30912856
Looser V. Bruhlmann B. Bumbak F. Stenger C. Costa M. Camattari A. Fotiadis D. Kovar K. Cultivation strategies to enhance productivity of Pichia pastoris: A review Biotechnol. Adv. 2015 33 1177 1193 26027890 10.1016/j.biotechadv.2015.05.008
Canales C. Altamirano C. Berrios J. The growth of Pichia pastoris Mut+ on methanol–glycerol mixtures fits to interactive dual-limited kinetics: Model development and application to optimised fed-batch operation for heterologous protein production Bioprocess Biosyst. Eng. 2018 41 1827 1838 10.1007/s00449-018-2005-1
van Bodegom P. Microbial Maintenance: A Critical Review on Its Quantification Microb. Ecol. 2007 53 513 523 10.1007/s00248-006-9049-5
Pirt S.J. Maintenance energy: A general model for energy-limited and energy-sufficient growth Arch. Microbiol. 1982 133 300 302 10.1007/BF00521294
Velastegui E. Theron C. Berrios J. Fickers P. Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris Yeast 2019 36 297 304 10.1002/yea.3381
Solà A. Jouhten P. Maaheimo H. Sánchez-Ferrando F. Szyperski T. Ferrer P. Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates Microbiology 2007 153 Pt 1 281 290 10.1099/mic.0.29263-0
Çelik E. Çalık P. Oliver S.G. Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rate Biotechnol. Bioeng. 2010 105 317 329 10.1002/bit.22543
Unrean P. Pathway analysis of Pichia pastoris to elucidate methanol metabolism and its regulation for production of recombinant proteins Biotechnol. Prog. 2014 30 28 37 10.1002/btpr.1855 24376216
Zepeda A.B. Figueroa C.A. Abdalla D.S. Maranhão A.Q. Ulloa P.H. Pessoa A. Jr. Farías J.G. HSF-1, HIF-1and HSP90 expression on recombinant Pichia pastoris under fed-batch fermentation Braz. J. Microbiol. 2014 45 485 490 10.1590/S1517-83822014000200015 25242931
Jordà J. de Jesus S.S. Peltier S. Ferrer P. Albiol J. Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids New Biotechnol. 2014 31 120 132 10.1016/j.nbt.2013.06.007 23845285
Lakshmanan M. Koh G. Chung B.K.S. Lee D.-Y. Software applications for flux balance analysis Brief. Bioinform. 2014 15 108 122 10.1093/bib/bbs069 23131418
Sillaparassamee O. Chinwetkitvanich S. Kanchanasuta S. Pisutpaisal N. Champreda V. Metabolic flux analysis on succinic acid production from crude glycerol by Actinobacillus succinogenes Biomass Convers. Biorefin. 2021 10.1007/s13399-021-01837-8
Lee S.Y. Lee D.Y. Hong S.H. Kim T.Y. Yun H. Oh Y.G. Park S. MetaFluxNet, a program package for metabolic pathway construction and analysis, and its use in large-scale metabolic flux analysis of Escherichia coli Genome Inform. 2003 14 23 33
Sánchez C.E.G. Sáez R.G.T. Comparison and analysis of objective functions in flux balance analysis Biotechnol. Prog. 2014 30 985 991 10.1002/btpr.1949
Bideaux C. Montheard J. Cameleyre X. Molina-Jouve C. Alfenore S. Metabolic flux analysis model for optimizing xylose conversion into ethanol by the natural C5-fermenting yeast Candida shehatae Appl. Microbiol. Biotechnol. 2016 100 1489 1499 10.1007/s00253-015-7085-0
Sibirny A.A. Ubiyvovk V.M. Gonchar M.V. Titorenko V.I. Voronovsky A.Y. Kapultsevich Y.G. Bliznik K.M. Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth Arch. Microbiol. 1990 154 566 575 10.1007/BF00248838
Tyurin O.V. Kozlov D.G. Deletion of the FLD gene in methylotrophic yeasts Komagataella phaffii and Komagataella kurtzmanii results in enhanced induction of the AOX1 promoter in response to either methanol or formate Microbiology 2015 84 408 411 10.1134/S0026261715030212
Shen S. Sulter G. Jeffries T.W. Cregg J.M. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris Gene 1998 216 93 102 10.1016/S0378-1119(98)00315-1
Yurimoto H. Lee B. Yasuda F. Sakai Y. Kato N. Alcohol dehydrogenases that catalyse methyl formate synthesis participate in formaldehyde detoxification in the methylotrophic yeast Candida boidinii Yeast 2004 21 341 350 10.1002/yea.1101 15042594
Sakai Y. Murdanoto A.P. Sembiring L. Tani Y. Kato N. A novel formaldehyde oxidation pathway in methylotrophic yeasts: Methylformate as a possible intermediate FEMS Microbiol. Lett. 1995 127 229 234 7758937 10.1111/j.1574-6968.1995.tb07478.x
Tomàs-Gamisans M. Ferrer P. Albiol J. Fine-tuning the P. pastoris iMT1026 genome-scale metabolic model for improved prediction of growth on methanol or glycerol as sole carbon sources Microb. Biotechnol. 2018 11 224 237 10.1111/1751-7915.12871
Ye R. Huang M. Lu H. Qian J. Lin W. Chu J. Zhuang Y. Zhang S. Comprehensive reconstruction and evaluation of Pichia pastoris genome-scale metabolic model that accounts for 1243 ORFs Bioresour. Bioprocess. 2017 4 22 10.1186/s40643-017-0152-x 28546903
Yang N.J. Hinner M.J. Getting Across the Cell Membrane: An Overview for Small Molecules, Peptides, and Proteins Methods Mol. Biol. 2015 1266 29 53 10.1007/978-1-4939-2272-7_3
Barrigon J.M. Valero F. Montesinos J.L. A macrokinetic model-based comparative meta-analysis of recombinant protein production by Pichia pastoris under AOX1 promoter Biotechnol. Bioeng. 2015 112 1132 1145 10.1002/bit.25518
Canales C. Altamirano C. Berrios J. Effect of dilution rate and methanol-glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture Biotechnol. Prog. 2015 31 707 714 10.1002/btpr.2069
Fukuoka H. Kawase T. Oku M. Yurimoto H. Sakai Y. Hayakawa T. Nakagawa T. Peroxisomal Fba2p and Tal2p complementally function in the rearrangement pathway for xylulose 5-phosphate in the methylotrophic yeast Pichia pastoris J. Biosci. Bioeng. 2019 128 33 38 10.1016/j.jbiosc.2019.01.008
Cai H.-L. Doi R. Shimada M. Hayakawa T. Nakagawa T. Metabolic regulation adapting to high methanol environment in the methylotrophic yeast Ogataea methanolica Microb. Biotechnol. 2021 14 1512 1524 10.1111/1751-7915.13811
