Gellissen, Gerd

Veenhuis, Marten

English

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   University of GroningenThe methylotrophic yeast Hansenula polymorphaGellissen, Gerd; Veenhuis, MartenPublished in:EPRINTS-BOOK-TITLEIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2001Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Gellissen, G., & Veenhuis, M. (2001). The methylotrophic yeast Hansenula polymorpha: its use infundamental research and as a cell factory. In EPRINTS-BOOK-TITLE University of Groningen, GroningenBiomolecular Sciences and Biotechnology Institute (GBB).CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 20-06-2022Meeting ReportThe methylotrophic yeast Hansenulapolymorpha: its use in fundamental researchand as a cell factoryGerd Gellissen1* and Marten Veenhuis21 Rhein Biotech GmbH, Eichsfelder Strasse 11, 40595 DuÈsseldorf, Germany2 Laboratory of Eukaryotic Microbiology, GBB, University of Groningen, Postbox 14, 9750 AA Haren, The Netherlands*Correspondence to:G. Gellissen, Rhein BiotechGmbH, Eichsfelder Strasse 11,40595 DuÈsseldorf, Germany.E-mail: gellissen_rheinbiotech@compuserve.comIntroductionDuring the last decade methylotrophic yeasts havegained increasing interest, both for fundamental(van der Klei and Veenhuis, 1996) and appliedpurposes (van Dijk et al., 2000; Gellissen, 2000).The special value of these yeasts (mainly Pichiapastoris and Hansenula polymorpha) in fundamentalresearch is undoubtedly related to studies on theprinciples of peroxisome homeostasis (biogenesis vs.degradation). In particular, the morphologicalfeatures of peroxisome development and selectivedegradation are more strongly pronounced than inSaccharomyces cerevisiae, which makes it possibleto induce and follow the details in mutants moreeasily. In addition, peroxisome induction in baker'syeast is restricted to growth on oleate (and thusb-oxidation), whereas in the methylotrophic yeastsvarious peroxisomal metabolic pathways mayoccur, depending on the composition of thegrowth medium. These organisms also display thestrongest induction rates that may rise to a level atwhich up to 80% of the total cell volume is occupiedby peroxisomes.The extremely high expression rates necessary toachieve this drew the attention of biotechnologists,who studied the use of these organisms as host forthe production of valuable, heterologous proteins.Initially, P. pastoris was predominantly used; nowH. polymorpha is gaining more and more attention.The H. polymorpha system was shown to provide aremarkably versatile technology platform for het-erologous gene expression. The Hansenula toolboxnow includes a variety of host strains, new integra-tion sites and several novel strong promotersbeyond those derived from methanol metabolismgenes. In the ®rst H. polymorpha worldwide net-work (HPWN) conference, the recent developmentsin the use of this organism were discussed in a livelyfashion. This report highlights several superb pre-sentations of important, interesting and novelresearch.H. polymorpha as a model system infundamental researchIn her keynote address, Ida van der Klei (Gronin-gen) summarized the recent developments onperoxisome homeostasis in H. polymorpha(Figure 1). These studies are facilitated by the factthat detailed information is now available on thephysiology, biochemistry and ultrastructure of theorganisms, allowing the precise manipulation ofperoxisome development, function and selectiveturnover of the organelles and their enzyme content(van der Klei and Veenhuis, 1997). Compartmenta-lization of speci®c functions into organelles isbelieved to be advantageous in the control ofvarious types of cellular processes, e.g. by theYeastYeast 2001; 18: i±iii.Copyright # 2001 John Wiley & Sons, Ltd.creation of unique microenvironments with speci®c(bio)chemical properties. The reasons for the cell tocompartmentalise part of the key enzymes ofmethanol metabolism are now understood. Thishas shown to be essential to: (a) regulate thepartition of formaldehyde produced from methanolover assimilatory and dissimilatory pathways; and(b) to ensure that H2O2, produced as a side productfrom methanol oxidation, is metabolized by perox-isomal catalase. Leakage of H2O2 from the orga-nelles is not lethal (as is often believed) but itsmetabolism outside the peroxisome leads to severeenergetic disadvantages that would prevent growthon methanol. As with other organelles, extremelycomplex regulatory mechanisms for peroxisomegeneration and maintenance have evolved. Apartfrom this, the function of the organelles should becontinuously and rapidly adaptable to ensure thatthe cell directs its resources optimally towardsspeci®c functions. One of the important ways toachieve this is to precisely control the abundance ofthe organelles. This may be achieved by tightlycontrolling two contradictory processes: the devel-opment of the organelles and their selective degra-dation under conditions that these organelles are nolonger required for growth. Evidence was presentedthat these two processes converge at Pex14p, aprotein which was initially described to be essentialfor peroxisome biogenesis. This control machineryenables the organism to maintain at least onesmall peroxisome after a shift of cells tonon-methylotrophic conditions. The physiologicaladvantage of this mechanism is immediately clear,since it allows the cell to adapt rapidly to a newenvironment that requires new peroxisome func-tions.JoseÂ Siverio gave a summary report on aspects ofnitrate metabolism in H. polymorpha (GonzaÂ lezet al., 1999). S. Krappmann and G. Braus describedthe ®rst gene and gene product involved in thebiosynthesis of aromatic amino acids, the HARO7gene, encoding chorismate mutase (Krappmannet al., 2000).H. polymorpha as a cell factoryA strong focus of the conference was the biotech-nological application of Hansenula polymorpha.Several contributors to the conference describednew elements for the further improvement of theplatform technology based on this yeast. The rangeof elements presented included new promoters,integration sites and auxotrophy complementationmarkers. Applied examples included production ofrecombinant gelatins, HSA, technical enzymesand a report about the production processes forH. polymorpha-derived hepatitis B vaccines thatwere launched a few years ago.C. P. Hollenberg from DuÈsseldorf and H. A.Kang from S.K. Rhee's group in Korea providedkeynote addresses describing the platform technol-ogy based on this yeast species. Integration intoHARS sequences and methods for the integrationof heterologous DNA in anticipated copy numbersare especially attractive tools (Sohn et al., 1999a, b).Several novel promoter elements were presented:P. Sudbery (Shef®eld) reported on the strongconstitutive expression of sequences fused to thePMA1-promoter (Cox et al., 2000). M. Suckow(Rhein Biotech GmbH) described a novel promoterderived from the heat-inducible TPS1 (trehalose-6-phosphate synthase) gene that appears to be evenstronger than the traditional methanol-inducibleFMD promoter (Amuel et al., 2000). New hostsbased on strains with a disrupted ARO7 geneexhibiting a tyrx phenotype were also described(Krappmann et al., 2000).Product and strain developments revealedinteresting new features of the H. polymorphasystem. E. de Bruin and F. de Wolf reported ontheir feasibility study for the production of recom-Figure 1. H. polymorpha cell, grown in a methanol-limitedchemostat, crowded with peroxisomesii G. Gellissen and M. VeenhuisCopyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: i±iii.binant collagens in this yeast. Collagens are derivedfrom gelatins by post-translational prolyl-4 hydro-xylation. This modi®cation could be accomplishedin other yeast systems by co-expression of arespective gene. The surprising ®nding of nativecollagen-like H. polymorpha proteins containing4-hydroxyproline suggests the existence of a suitableendogenous hydroxylase gene, which could makethe production of heterologous collagens moreef®cient (de Bruin et al., 2000).T. McMullin (BTR, USA) presented his group'sefforts in the development of strains for theproduction of a fungal galactose oxidase. The datademonstrated a complex relationship of gene copynumber and the accumulation of the gene product,suggesting that using gene copy number as apredictor of product accumulation may not alwaysbe accurate.S.-J. Ahn (GCVC, Korea) presented a detailedreport on the production process for theH. polymorpha-derived hepatitis B vaccine. GCVCreceived the status of a pre-quali®ed supplier forthis vaccine from the World Health Organization,making H. polymorpha the only eukaryotic micro-bial expression system beside S. cerevisiae to beapplied for the production of a launched pharma-ceutical (Schaefer et al., 2000).The conference was also the initiation of theH. polymorpha Worldwide Network, aimed atproviding a platform for information exchangebetween scientists working with this yeast. Thenext conference is scheduled for 2002, and a webpage is currently in preparation.For further information, please contact G.Gellissen (gellissen_rheinbiotech@compuserve.com)or M. Veenhuis (m.veenhuis@biol.rug.nl)AcknowledgementsWe thank the participants of the meeting for their valuablecontributions to the meeting and for allowing us to quotetheir unpublished results. We thank Rhein Biotech GmbH,DuÈsseldorf, for ®nancial and logistic support for thisconference.ReferencesAmuel C, Gellissen G, Hollenberg CP, Suckow M. 2000.Analysis of heat shock promoters in Hansenula polymorpha:the TPS1 promoter, a novel element for heterologous geneexpression. Biotechnol Bioproc Eng 5: 247±252.de Bruin EC, de Wolf FA, Laane NC. 2000. Expression andsecretion of human alpha-1 (I) procollagen fragment byHansenula polymorpha as compared to Pichia pastoris.Enzyme Microb Technol 26: 640±644.Cox H, Mead D, Sudbery P, Eland M, Mannazzu I, Evans L.2000. Constitutive expression of recombinant proteins in themethylotrophic yeast Hansenula polymorpha using the PMA1promoter. Yeast 16: 1191±1203.van Dijk R, Faber KN, Kiel JAWK, Veenhuis M, van der Klei I.2000. The methylotrophic yeast Hansenula polymorpha:a versatile cell factory. Enzyme Microb Technol 26:793±800.Gellissen G. 2000. Heterologous protein production in methylo-trophic yeasts. Appl Microbiol Biotechnol 54: 741±750.GonzaÂlez C, Perdomo G, Tejera P, Brito N, Siverio JM. 1999.One-step, PCR-mediated gene disruption in the yeast Hanse-nula polymorpha. Yeast 15: 1323±1329.van der Klei IJ, Veenhuis M. 1996. Peroxisome biogenesis in theyeast Hansenula polymorpha: a structural and functionalanalysis. Ann NY Acad Sci 804: 47±59.van der Klei IJ, Veenhuis M. 1997. Yeast peroxisomes: functionand biogenesis of a versatile cell organelle. Trends Microbiol5(12): 502±509.Krappmann S, Pries R, Gellissen G, Hiller M, Braus GH. 2000.HARO7 encodes chorismate mutase of the methylotrophicyeast Hansenula polymorpha and is derepressed upon methanolutilization. J Bacteriol 182: 4188±4197.Schaefer S, Piontek M, Ahn S-J, Papendieck A, Janowicz ZA,Gellissen G. 2001. Hepatitis B vaccines±characterization of theviral disease and vaccine production in the methylotrophicyeast, Hansenula polymorpha. In Novel Therapeutic Proteins±Selected Case Studies, Dembowsky K, Stadler P (eds). Wiley-VCH: Weinheim; 245±274.Sohn JH, Choi ES, Kang HA, Rhee JS, Rhee SK. 1999a.A family of telomere-associated autonomously replicatingsequences and their functions in targeted recombination inHansenula polymorpha DL-I. J Bacteriol 181: 1005±1013.Sohn JH, Choi ES, Kang HA et al. 1999b. A dominant selectionsystem designed for copy-number-controlled gene integrationin Hansenula polymorpha DL-I. Appl Microbiol Biotechnol 51:800±807.Hansenula polymorpha iiiCopyright # 2001 John Wiley & Sons, Ltd. Yeast 2001; 18: i±iii.

The methylotrophic yeast Hansenula polymorpha:its use in fundamental research and as a cell factory

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The methylotrophic yeast Hansenula polymorpha:its use in fundamental research and as a cell factory

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