Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment
© Stoeckmann et al; licensee BioMed Central Ltd. 2009
Received: 07 January 2009
Accepted: 15 April 2009
Published: 15 April 2009
A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.
Hansenula polymorpha and Arxula adeninivorans and their competitive environment
In the last three decades a wide range of recombinant proteins, especially pharmaceuticals, have been produced based on heterologous gene expression in bacterial organisms, mammalian cells and several yeasts and fungi [1–3]. Production processes had to be developed that employ platforms which meet both, the demand for efficient mass production and criteria of safety and authenticity of the produced compounds. In this respect yeasts offer considerable advantages over alternative microbial and mammalian cell systems in providing low-cost screening and production systems for authentically processed and modified proteins. The organisms meet safety prerequisites in that they do not harbour pyrogens, pathogens or viral inclusions [4, 5]. Recent engineering of yeast hosts with the capability to add humanized N-glycans of the intermediate mannose type  or even the complex type  provides the option to produce biopharmaceuticals with human protein modifications. The recognition of yeasts as attractive expression platforms for biopharmaceuticals is met by genome analysis of an increasing number of yeast species, among others that of Saccharomyces cerevisiae  and Hansenula polymorpha .
As a consequence some early examples of FDA-approved biopharmaceuticals like insulin  and hepatitis B vaccines [10, 11] have been produced in the baker's yeast S. cerevisiae. However, certain limitations and drawbacks are encountered when using this system: S. cerevisiae tends to hyperglycosylate recombinant proteins; N-linked carbohydrate chains are terminated by mannose attached to the chain via an α1,3 bond, which is considered to be allergenic. The limited carbon source utilization imposes restrictions on the design of fermentation processes; due to the preferential use of episomal vectors instabilities of recombinant strains and as a result batch inconsistencies of production runs are of major concern .
Therefore an increasing number of alternative yeast systems have been defined that can potentially overcome the described limitations of the traditional baker's yeast. The availability of a wide-range yeast vector system (CoMed™) enables the assessment of several yeasts in parallel for their capability to produce a particular protein in desired quality with a single vector to identify an optimal host at the beginning of a product and process development. For expression control the wide-range vector contains a constitutive TEF1 promoter derived from various sources that is active in all yeast species analyzed so far. If needed this promoter element can easily be substituted during further strain development by a promoter optimal for the defined platform [, Additional file 1]. Out of the plethora of addressable species we describe in this article methylotrophic H. polymorpha, a recognized producer of biopharmaceuticals and other recombinant proteins, and dimorphic Arxula adeninivorans, a novel platform that has yet to establish itself for industrial applications. First experiments indicate that screening and fermentation conditions based on minimal SYN6 medium (SYN6) with glucose supplementation as described in this article can also be applied to yeast platforms others than H. polymorpha and A. adeninivorans.
The range of biotechnologically applied methylotrophic yeasts furthermore includes Candida boidinii, Pichia methanolica, and Pichia pastoris . In all instances most examples of heterologous gene expression are linked to strong and adjustable promoters derived from genes of the methanol utilization pathway [12, 17], most commonly the elements derived from the alcohol oxidase genes, namely AOX1 from P. pastoris [16–18], MOX from H. polymorpha [14, 17]; AOD1 from C. boidinii [16, 17, 19] and AUG1 (now designated MOD1) from P. methanolica [16, 17, 20]. In H. polymorpha the FMD (formate dehydrogenase) promoter, derived from another methanol utilization pathway gene of similar regulation, has found preferential application to established industrial processes [14, 16].
A. adeninivorans (Blastobotrys adeninivorans) is a yeast with unusual characteristics. It is a dimorphic species and can utilize adenine, xanthine, uric acid, putrescine and n-alkylamines as carbon, nitrogen or energy sources in addition to glucose. Like H. polymorpha it is a nitrate-assimilating, thermo- and osmotolerant organism. A distinctive feature is a temperature-dependent dimorphism with mycelial structures formed at temperatures above 42°C [5, 12] [Fig. 1b, c]. For Fe(II)-oxidase Afet3p, O-glycosylation was observed to be restricted to the budding cell status . It remains to be shown whether this differential O-glycosylation pattern in correlation to the morphological status is also present in recombinant and other host proteins. Again, several strains have been identified after its first description as Trichosporon adeninovorans . Most of the research and the biotechnological applications have been performed with strain LS3 (PAR-4), isolated in Sibiria by Kapultsevich, and a range of auxotrophic mutants have been generated [5, 12]. Strain 135 is a mutant that forms mycelial structures at 30°C . Recently auxotrophic host strains for heterologous gene expression have been generated based on strains CBS7350 and CBS1738 (see Tab. 1). So far, no industrial A. adeninivorans-based process exists. For expression and fermentation studies on a laboratory scale heterologous genes were mostly expressed under control of TEF1, a constitutive A. adeninivorans-derived promoter of appropriate strength .
For description we selected established H. polymorpha-based processes with strains expressing a heterologous gene under control of the adjustable FMD and MOX promoters and A. adeninivorans-based lab scale processes with strains expressing a heterologous gene under control of the constitutive A. adeninivorans-derived TEF1 promoter, with culturing conditions that can possibly be applied to the assessment of other yeasts with constitutive heterologous gene expression. Micrographs of the two selected platforms are shown in Fig. 1.
H. polymorpha-based processes under control of MOX and FMD promoters – a re-assessment
MOX and FMD are genes encoding enzymes of the methanol utilization pathway that is shared by all methylotrophic yeasts. The enzyme components of this pathway and their control have been reviewed extensively in the recent past [4, 17, 19]. The genes of this pathway are described to be tightly regulated; they are highly repressed in the presence of non-limiting concentrations of glucose and strongly induced if methanol is used as a carbon source . Methylotrophic growth is furthermore accompanied by a massive proliferation of peroxisomes in which several methanol-metabolizing enzymes are compartmentalized [19, 24]. However, it soon became evident that activation of methanol pathway promoters did not depend on the presence of methanol in H. polymorpha in contrast to the situation in the other methylotrophs . For all other methylotrophic yeast species an inductive activation of such promoters has been stated that is strictly dependent on the presence of methanol . As a consequence several H. polymorpha-based industrial fermentation processes have been defined that lean on glucose or glycerol supplementation in suitable concentrations to a culture broth without any methanol additions [1, 25, 26].
The distinct feature of the H. polymorpha-derived methanol pathway promoters was elucidated, when new tools of genomics and postgenomic analysis became available. After sequencing the entire genome of strain CBS4732  a cDNA microarray was constructed that allowed comprehensive gene expression profiling [27, 28]. When analyzing the transcriptome of H. polymorpha strains of glucose-supplemented growth and after transition to methanol-supplemented growth it became evident that the methanol dissimilation genes including MOX and FMD are activated by de-repression upon carbon source limitation and depletion and not upon induction by methanol. In contrast genes of peroxisome biogenesis and proliferation are induced by methanol [14, 15].
With respect to these findings, recombinant H. polymorpha strains expressing a GFP reporter gene under control of the FMD promoter were screened applying glucose- or glycerol-supplemented media to strain culturing.
An early example for a glycerol starvation process is the production process for hirudin [33–35] [Fig. 3a]. As in all other described processes expression vectors were constructed to transform the uracil-auxotrophic strain RB11, like strain MedHp1 (Tab. 1) a derivative of strain LR9 [36–38]. The vectors contain an expression cassette with a hirudin sequence under control of the MOX promoter. For secretion it was fused to a secretion leader, derived from the pheromone precursor MFα1p . Fermentation on a 35 liter scale was carried out at 30°C in SYN6 at pH 5. It was started with 30 g/L of glycerol. After consumption of the carbon source after 35 hours a feed was initiated that added glycerol by a pO2-controlled feeding device. Hirudin production started after some 20 hours when the MOX-promoter was activated by de-repression under glycerol limitation. The subsequent feeding conditions supported the de-repressed status of the promoter by maintaining glycerol concentrations between 0.5 and 3.0 g/L. These feeding conditions resulted in an increasing accumulation of the product in the medium [33, 34, 39] [Fig. 3a].
Similar fermentation conditions were applied to culturing of production strains for the anticoagulant saratin [35, 39] or for aprotinin , now applying the FMD promoter to expression control. This fermentation design was modified when developing a production process for the cytokine IFNalpha-2a [40, 41]. IFNalpha-2a forms a disulfide bond between amino acids Cys1 and Cys98. Bond formation of the first amino acid Cys1 of the mature sequence provides a steric hindrance for correct maturation when processed from an MFα1/IFNalpha-2a precursor. Accordingly a large share of secreted recombinant hirudin consisted of incorrectly processed molecules with N-terminal extensions. This could be overcome by co-production of the processing enzyme Kex2p, however at the expense of a more pronounced proteolytic degradation. To minimize this degradation pH was lowered from pH 5 (as applied to standard fermentations) to pH 2–3. The glycerol starvation conditions for FMD promoter de-repression remained unchanged.
For the production of phytase an extremely efficient production process has been developed. In this process all steps and components of the process followed a rationale of efficiency and cost-effectiveness. This rationale provoked an assessment of glucose as sole carbon source for fermentation . In a fermentation of a phytase production strain with FMD-controlled expression on 2000 L scale glucose was supplemented as 20 g/L. Upon depletion, a glucose-limiting feed was initiated that added the carbon source with a stepwise increasing feeding rate in correlation to the cell mass. In this glucose starvation process a final yield of 13.5 g/L phytase was observed [42, 43] [Fig. 3b].
The only established industrial fermentation process with methanol supplementation is that for the production of the hepatitis B surface antigen HBsAg, the first biopharmaceutical produced in H. polymorpha. Several processes for this vaccine have been described that are based on both, MOX or FMD-controlled expression . In Fig. 3c a typical fermentation process is schematically shown. The batch phase and a first fed-batch phase is similar to the examples of glycerol starvation described before [Fig. 3a]. In contrast a mixture of glycerol and methanol is fed during the last hours of fermentation. Obviously this results in an inductive increase of HBsAg production. However, in view of the transcriptome profiling it seems that methanol supplementation does not induce the FMD-controlled expression of the heterologous antigen gene, but it induces membrane proliferation. As the HBsAg is produced as particles with the recombinant antigen inserted into host-derived membranes, methanol is considered to provide a balanced co-production of both particle components in high titers [10, 11].
The selected process examples demonstrate the possibility to develop efficient screening and fermentation processes for strains with MOX- or FMD-driven heterologous gene expression without methanol supplementation to a medium.
Culturing of Arxula adeninivorans strains
For A. adeninivorans industrial process parameters have not been defined yet and mainly cultivations on a shake flask scale have been carried out so far. Most of the current expression studies are based on wild type strain LS3  or its leucine-auxotrophic derivative G1211. Additional strains and leucine-auxotrophic mutants thereof have been established more recently (see Tab. 1).
Acid phosphatase production was characterized in fermentations of both strain LS3 and a recombinant strain expressing the APHO1 gene under control of the strong TEF1 promoter . Using the Plackett-Burman design three variables (pH, sucrose concentrations, and peptone concentration) were optimized for medium composition, a roughly four times enhancement was observed in media containing 39 g/L sucrose and 16 g/L peptone at pH 3.8 .
Shake flask cultures of A. adeninivorans strains were analyzed using a device for online measurement of the respiration rates (RAMOS, r espiratory a ctivity m onitoring s ystem) [47, 48]. This device had previously been applied to the analysis of H. polymorpha cultures and to alternative platforms [49–52].
In fermentations of strain LS3 in MES-buffered YMM* the respiration rate exponentially increased followed by a linear increase presumably indicating a nutrient deficiency. After 24 hours, respiration rate dropped upon glucose depletion. [Fig. 4, grey circles].
Subsequently glucose-supplemented SYN6 was assessed for applicability to shake flask cultures of A. adeninivorans. The high nutrient concentrations of standard SYN6 remained unchanged. Again, the pH of SYN6 had to be buffered with MES (SYN6-MES, see  for detailed description) for pH stabilization between 6.4 and 5.3. Fig. 4, open circles shows the respiration rate of a shake flask culture with strain LS3 in SYN6-MES. The described culture course adverts to the non-limited growth of A. adeninivorans in the respective shake flask culture. Thus, favourable non-limiting growth conditions for A. adeninivorans in shake flasks were proven to be developed.
Additionally suitable fed-batch conditions for high cell density fermentations (HCDF) in SYN6 were found. These conditions were assessed for culturing a recombinant A. adeninivorans strain producing phytase under control of the TEF1 promoter [5, 53]. During the feeding under glucose-limitation phytase was secreted to maximal titres of ca. 900 FTU/mL (one FTU equates to the phytase amount liberating 1 μmol of inorganic phosphate per minute at pH 5.5 and 37°C) . Thus, the growth conditions defined for shake flask cultures and HCDF of A. adeninivorans wild type strain LS3, proved to be applicable for the phytase-producing recombinant A. adeninivorans strain. Finally the phytase-producing strain was cultured under pressurized conditions in a 50 L STR, again using a SYN6-derived medium for culturing. During the fed-batch phase the reactor pressure was increased stepwise up to 5 bar. After 42 h cells had grown up to 224 g/L. Phytase amount increased up to 10 × 106 FTU. Fermentations under pressurized conditions may result in increased product yields and shorter fermentation time .
The conditions of culturing can potentially be applied to screening and culturing of other yeasts expressing a foreign gene under control of a constitutive TEF1 promoter – a key element of the CoMed™ system described in the following section.
The CoMed™ system
It is desirable that the range of yeasts addressed in parallel can be also assessed in parallel for optimal performance in a given case. First experiments indicate that SYN6 and derivatives thereof are suitable minimal media for yeasts others than A. adeninivorans and H. polymorpha. The general use of the constitutive TEF1 promoter is expected to ensure screening and fermentation conditions similar to those described for H. polymorpha and A. adeninivorans.
HA Kang was supported by a grant from the Korean Ministry of Science and Technology (Microbial Genomics and Applications Research and Development Program).
The content of this review has been presented at the conference Biotech2008: Biopharmaceuticals: Why yeasts? Wädenswil, Switzerland. May 23 2008.
- Gellissen G: Production of recombinant proteins: Novel microbial and eukaryotic expression systems. 2005, Weinheim: Wiley-VCHGoogle Scholar
- Melmer G: Biopharmaceuticals and the industrial environment. Production of recombinant proteins: novel microbial and eukaryotic expression systems. Edited by: Gellissen G. 2005, 361-383. Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Yin J, Li G, Reu X, Herler G: Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol. 2007, 127: 335-347.View ArticleGoogle Scholar
- Gellissen G, Kunze G, Gaillardin C, Cregg JM, Berardi E, Veenhuis M, Klei van der I: New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and dimorphic Arxula adeninivorans and Yarrowia lipolytica – a comparison. FEMS Yeast Res. 2005, 5: 1079-1096.View ArticleGoogle Scholar
- Böer E, Gellissen G, Kunze G:Arxula adeninivorans. Production of recombinant proteins: novel microbial and eukaryotic expression systems. Edited by: Gellissen G. 2005, 89-110. Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Kim MW, Kim EJ, Kim JY, Park JS, Oh DB, Shimma JI, Chiba Y, Jigami Y, Rhee SK, Kang HA: Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation. J Biol Chem. 2006, 281: 6261-6272.View ArticleGoogle Scholar
- Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, Bobrowicz P, Stadheim TA, Li H, Choi BK, Hopkins D, Wischnewski H, Roser J, Mitchell T, Strawbridge RR, Hoopes J, Wildt S, Gerngross TU: Humanization of yeast to produce complex terminally sialylated glycoproteins. Science. 2006, 313: 1441-1443.View ArticleGoogle Scholar
- Goffeau A, Barrell RG, Busey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG: Life with 6000 genes. Science. 1996, 274: 563-567.View ArticleGoogle Scholar
- Ramezani-Rad M, Hollenberg CP, Lauber J, Wedler H, Griess E, Wagner C, Albermann K, Hani J, Piontek M, Dahlems U, Gellissen G: The Hansenula polymorpha (strain CBS4732) genome – sequencing and analysis. FEMS Yeast Res. 2003, 4: 207-215.View ArticleGoogle Scholar
- Brocke P, Schaefer S, Melber K, Jenzelewski V, Mueller F, Dahlems U, Bartelsen O, Park KN, Janowicz ZA, Gellissen G: Hepatitis B vaccines – disease characterization and vaccine production. Production of recombinant proteins: novel microbial and eukaryotic expression systems. Edited by: Gellissen G. 2005, 319-60. Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Melmer G, Kunze G, Gellissen G: Recombinant vaccine production in yeast. Biopharm Intern January Suppl. 2008, 8-13.Google Scholar
- Böer E, Steinborn G, Gellissen G, Kunze G: Production of interleukin-6 in Arxula adeninivorans, Hansenula polymorpha and Saccharomyces cerevisiae by applying a wide-range yeast vector (CoMed™) system to simultaneous comparative assessment. FEMS Yeast Res. 2007, 7: 1181-1187.View ArticleGoogle Scholar
- Steinborn G, Böer E, Scholz A, Tag K, Kunze G, Gellissen G: Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microbial Cell Factories. 2006, 5: 33-View ArticleGoogle Scholar
- Kang HA, Gellissen G:Hansenula polymorpha. Production of recombinant proteins: novel microbial and eukaryotic expression systems. Edited by: Gellissen G. 2005, 111-142. Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Kunze G, Kang HA, Gellissen G: Hansenula polymorpha – biology and applications. Yeast Biotechnology: Diversity and applications. Edited by: Satyaranayana T, Kunze G. Berlin: SpringerGoogle Scholar
- Gellissen G: Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol. 2000, 54: 741-750.View ArticleGoogle Scholar
- Hartner FS, Glieder A: Regulation of methanol utilisation pathway genes in yeasts. Microbial Cell Factories. 2006, 5: 39-View ArticleGoogle Scholar
- Tschopp JF, Brust TF, Cregg JM, Stillman CA, Gingeras TR: Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 1987, 15: 3859-3876.View ArticleGoogle Scholar
- Yurimoto H, Sakai Y, Kato N: Methanol metabolism. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 61-75. Weinheim: Wiley-VCHGoogle Scholar
- Nakagawa T, Inagaki A, Ito T, Fujimura S, Mijaji T, Yurimoto H, Kato N, Sakai Y, Tomizuka N: Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica. Yeast. 2006, 23: 15-22.View ArticleGoogle Scholar
- Wartmann T, Stephan UW, Bube I, Böer E, Melzer M, Manteuffel R, Stoltenburg R, Guengerich L, Gellissen G, Kunze G: Post-translational modifications of the AFET3 gene product – a component of the iron transport system in budding cells and mycelia of the yeast Arxula adeninivorans. Yeast. 2002, 19: 849-862.View ArticleGoogle Scholar
- Middelhoven WJ, Hoogkamer-Te Niet C, Kreger Van Rij NWJ: Trichosporon adeninovorans sp. nov., a yeast species utilizing adenine, xanthine, uric acid, putrescine and primary n-alkylamines as sole source of carbon, nitrogen and energy. Antonie van Leeuwenhoek. 1984, 50: 369-378.View ArticleGoogle Scholar
- Wartmann T, Erdmann J, Kunze I, Kunze G: Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3. Arch Microbiol. 2000, 173: 253-261.View ArticleGoogle Scholar
- Klei van der IJ, Veenhuis M: Hansenula polymorpha – a versatile model in peroxisome research. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 76-94. Weinheim: Wiley-VCHGoogle Scholar
- Hellwig S, Stöckmann C, Gellissen G, Büchs J: Comparative fermentation. Production of recombinant proteins: novel microbial and eukaryotic expression systems. Edited by: Gellissen G. 2005, 287-317. Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Gellissen G: Hansenula polymorpha – biology and applications. 2002, Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Oh KS, Kwon O, Oh YW, Sohn MJ, Jung S, Kim YK, Kim MG, Rhee SK, Gellissen G, Kang HA: Fabrication of a partial genome microarray of the methylotrophic yeast Hansenula polymorpha: Optimization and evaluation for transcript profiling. J Microbiol Biotechnol. 2004, 14: 1239-1248.Google Scholar
- Park JN, Sohn MJ, Oh DB, Kwon O, Rhee SK, Hur CG, Lee SY, Gellissen G, Kang HA: Identification of the cadmium-inducible Hansenula polymorpha SEO1 gene promoter by transcriptome analysis and its application to whole-cell heavy-metal detection systems. Appl Environm Microbiol. 2007, 73: 5990-6000.View ArticleGoogle Scholar
- Berrow NS, Büssow K, Coutard B, Diprose J, Ekberg M, Folkers GE, Levy N, Lieu V, Owens RJ, Peleg Y, Pinaglia C, Quevillon-Cheruel S, Salim L, Scheich C, Vinventelli R, Busso D: Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr D Biol Crystallogr. 2006, 62: 1218-1226.View ArticleGoogle Scholar
- Jeude M, Dittrich B, Niederschulte H, Anderlei T, Knocke C, Klee D, Büchs J: Fed-batch mode in shake flasks by slow-release technique. Biotechnol Bioeng. 2006, 95: 433-445.View ArticleGoogle Scholar
- Amuel C, Gellissen G, Hollenberg CP, Suckow M: Analysis of heat shock promoters in Hansenula polymorpha: the TPS 1 promoter, a novel element for heterologous gene expression. Biotechnol Bioprocess Eng. 2000, 5: 247-252.View ArticleGoogle Scholar
- Jenzelewski V: Fermentation and primary product recovery. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 156-174. Weinheim: Wiley-VCHGoogle Scholar
- Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G: High-level production of hirudin by Hansenula polymorpha – authentic processing of three different preprohirudins. Appl Microbiol Biotechnol. 1995, 44: 377-385.View ArticleGoogle Scholar
- Avgerinos GC, Turner BG, Gorelick KJ, Papendieck A, Weydemann U, Gellissen G: Production and preclinical analysis of H. polymorpha-derived PEG-hirudin. Sem Thromb Hemostas. 2001, 27: 357-371.View ArticleGoogle Scholar
- Barnes CS, Krafft B, Frech M, Hoffmann UR, Papendieck A, Dahlems U, Gellissen G, Hoylarts MF: Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen. Semin Thromb Hemost. 2001, 27: 337-347.View ArticleGoogle Scholar
- Roggenkamp R, Hansen H, Eckart M, Janowicz ZA, Hollenberg CP: Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet. 1988, 202: 302-308.View ArticleGoogle Scholar
- Zurek C, Kubis E, Keup P, Hörlein D, Beunink J, Thömmes J, Kula MR, Hollenberg CP, Gellissen G: Production of two aprotinin variants in Hansenula polymorpha. Proc Biochem. 1996, 31: 679-689.View ArticleGoogle Scholar
- Suckow M, Gellissen G: The expression platform based on Hansenula polymorpha RB11 – history, status and perspectives. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 105-123. Weinheim: Wiley-VCHGoogle Scholar
- Bartelsen O, Barnes CS, Gellissen G: Production of anticoagulants in Hansenula polymorpha. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 211-228. Weinheim: Wiley-VCHGoogle Scholar
- Müller F, Tieke A, Waschk D, Mühle C, Müller F, Seigelchifer M, Pesce A, Jenzelewski V, Gellissen G: Production of IFNα-2a in Hansenula polymorpha. Proc Biochem. 2002, 38: 15-25.View ArticleGoogle Scholar
- Gellissen G, Müller F, Sieber H, Tieke A, Jenzelewski V, Degelmann A, Strasser AWM: Production of cytokines in Hansenula polymorpha. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 229-254. Weinheim: Wiley-VCHView ArticleGoogle Scholar
- Mayer AF, Hellmuth K, Schlieker H, Lopez-Ulibarri R, Oertel S, Dahlems U, Strasser AWM, van Loon APGM: An expression system matures: a highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha. Biotechnol Bioeng. 1999, 63: 373-381.View ArticleGoogle Scholar
- Papendieck A, Dahlems U, Gellissen G: Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha. Hansenula polymorpha – biology and applications. Edited by: Gellissen G. 2002, 255-271. Weinheim: Wiley-VCHGoogle Scholar
- Kunze G, Kunze I: Characterization of Arxula adeninivorans from different habitats. Antonie van Leeuwenhoek. 1994, 65: 29-34.View ArticleGoogle Scholar
- Kaur P, Lingner A, Singh B, Böer E, Polajeva J, Steinborn G, Bode R, Gellissen G, Satyanarayana T, Kunze G: APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity. A van Leeuwenhoek. 2007, 91: 45-55.View ArticleGoogle Scholar
- Minocha N, Kaur P, Satyanarayana T, Kunze G: Acid phosphatase production by recombinant Arxula adeninivorans. Appl Microbiol Biotechnol. 2007, 76: 387-393.View ArticleGoogle Scholar
- Anderlei T, Büchs J: Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem Eng J. 2001, 7: 157-162.View ArticleGoogle Scholar
- Anderlei T, Zang W, Papaspyrou M, Büchs J: Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochem Eng J. 2004, 17: 187-194.View ArticleGoogle Scholar
- Silberbach M, Maier B, Zimmermann M, Büchs J: Glucose oxidation by Gluconobacter oxydans: characterization in shaking flasks, scale-up and optimization of the pH profile. Appl Microbiol Biotechnol. 2003, 62: 92-98.View ArticleGoogle Scholar
- Stöckmann C, Losen M, Dahlems U, Knocke C, Gellissen G, Büchs J: Effect of oxygen supply on passaging, stabilisation and screening of recombinant H. polymorpha production strains in test tubes cultures. FEMS Yeast Res. 2003, 4: 195-205.View ArticleGoogle Scholar
- Stöckmann C, Maier U, Anderlei T, Knocke C, Gellissen G, Büchs J: The oxygen transfer rate as key parameter for the characterization of Hansenula polymorpha screening cultures. J Ind Microbiol Biotechnol. 2003, 30: 613-622.View ArticleGoogle Scholar
- Losen M, Froehlich B, Pohl M, Büchs J: Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures. Biotechnology Progress. 2004, 20: 1062-1068.View ArticleGoogle Scholar
- Rösel H, Kunze G: Cloning and characterization of a TEF1 gene for elongation factor 1α from the yeast Arxula adeninivorans. Curr Genet. 1995, 28: 360-366.View ArticleGoogle Scholar
- Knoll A, Bartsch S, Husemann B, Engel P, Schroer K, Ribeiro B, Stöckmann C, Seletzky J, Büchs J: High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank reactors. J Biotechnol. 2007, 132: 167-179.View ArticleGoogle Scholar
- Böer E, Steinborn G, Matros A, Mock HP, Gellissen G, Kunze G: Production of interleukin-6 in Arxula adeninivorans, Hansenula polymorpha and Saccharomyces cerevisiae by applying the wide-range yeast vector (CoMed™) system to simultaneous comparative assessment. FEMS Yeast Res. 2007, 5: 1181-1187.View ArticleGoogle Scholar
- Böer E, Steinborn G, Kunze G, Gellissen G: Yeast expression platforms. Appl Microbiol Biotechnol. 2007, 77: 13-523.View ArticleGoogle Scholar
- Steinborn G, Böer E, Kunze G, Gellissen G: Application of the CoMed™ system. Yeast Biotechnology: Diversity and applications. Edited by: Satyaranayana T, Kunze G. Berlin: SpringerGoogle Scholar
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