A Versatile Screening Tool for a Yeast Expression Platform

Why is a wide-range yeast expression vector system needed?

Yeasts include a great diversity of organisms that provide excellent hosts for the production of recombinant proteins. Some of the yeast-based expression platforms like the baker’s yeast Saccharomyces cerevisiae and the methylotrophs Hansenula polymorpha and Pichia pastoris are distinguished producers of launched biopharmaceuticals whereas other more recently defined systems like Yarrowia lipolytica or Arxula adeninivorans have yet to establish themselves as industrial standards. Yeasts offer a desired ease of genetic manipulation and rapid growth to high cell densities on inexpensive defined synthetic media. As eukaryotes, they are able to secrete foreign proteins and to perform multiple posttranslational modifications associated with the secretory pathway, thus producing even complex foreign proteins that are often identical or very similar to native products from plant or mammalian cells [1-3]. However differences in productivity, secretion or product modification has been observed among the different yeast platforms. The initial yeast expression platform is based on the baker’s yeast S. cerevisiae, the species of the longest tradition of human use.

Although successfully applied to the production of biopharmaceuticals like insulin or hepatitis B vaccines some important disadvantages became apparent, thereby restricting its general use. Glycoproteins are often over-glycosylated, and terminal mannose residues in N-linked glycans are added by an alpha-1,3 bond which is suspected to be allergenic [4,5]. Instead, non-allergenic terminal alpha-1,2 mannosylation is observed in the methylotrophs Hansenula polymorpha and Pichia pastoris [5,6]. In dimorphic Arxula adeninivorans the synthesis of O-glycosylated iron transport protein Fet3p is restricted to the budding yeast status, whereas cells of mycelial growth (present at temperatures above 42 °C) do not produce such structures. This may provide an option for differential modification of foreign proteins in an identical strain by applying different temperature conditions [7]. The content of proteolytic enzymes differs among yeasts: in case of recombinant hirudin-producing yeast strains the extent of degradation was found to be much higher in S. cerevisiae than in H. polymorpha [8].

In a recent comparative study it was shown that the cytokine IL-6 is correctly processed from a MFalpha1 leader/IL-6 fusion in A. adeninivorans, but that N-terminally truncated cytokines are secreted from H. polymorpha, P. pastoris and S. cerevisiae hosts [9,10]. Growth on glucose imposes limitations to the fermentation design for S. cerevisiae strains [11]. Most other yeast species can utilize a wider range of carbon sources thereby providing attractive strong promoter elements derived from the various utilization pathways and as a consequence enabling various options for fermentation design. Production of particular hydrophobic proteins may only be feasible in certain hosts. The two methylotrophic species H. polymorpha and P. pastoris offer a distinct methanol and carbon source requirement for the activation of promoters derived from genes of the methanol utilization pathway [6,12]. These few examples illustrate the necessity of carefully assessing a range of fungal organisms before defining an expression platform.

In spite of favorable characteristics and advantages for specific products it is evident that no single yeast system is optimal for all proteins. Sometimes attempts fail completely to produce a heterologous protein, in other cases productivity or modifications are severely impaired, thereby preventing the development of a competitive production process or a marketable pharmaceutical. Predictions for a successful strain and process development can only be made to a certain extent and misjudgments cannot be excluded. The initial definition of a platform may result in costly time- and resource-consuming failures. It is therefore advisable to assess primarily several yeasts in parallel for criteria like appropriate protein processing or secretion in a given case. A vector that can be simultaneously targeted to the various platform candidates greatly facilitates such a comparison.

Design and essential elements of a wide-range expression vector

The design of a vector suited for a wide range of fungal organisms has to meet functionality for all platforms to be assessed. As such a plasmid has to contain a targeting element, a promoter that drives heterologous gene expression and a selection marker suitable for all test species.

Sequences of the rDNA encoding ribosomal RNAs provide targeting elements that are highly conserved among the various yeasts. The rDNAs consist of clusters of repeated identical units present in high copy number (between 30-50 in H. polymorpha [13,14] to 200 in S. cerevisiae [15]. They are readily accessible to all components required for an efficient transcription.

The conservation of rDNA sequences is restricted to sequences encoding the various rRNA species contained in a unit; the sequences of non-coding segments can be quite divergent. Therefore elements derived from coding sequences or elements containing extended segments of these conserved sequences have to be employed. Elements exclusively consisting of non-coding sequences are likely to function in a species-specific manner only. Recently rDNA sequences of A. adeninivorans and H. polymorpha with appropriate characteristics have been defined as targeting elements composed of both, coding and potential regulatory non-coding sequences [16].

For selection, a range of dominant selection markers like the E. coli-derived hph gene conferring resistance against hygromycin [17,18] or genes of different sources that complement auxotrophies of respective host strains can be selected. Examples are the A. adeninivorans-derived LEU2 gene [19] or the H. polymorpha- or S. cerevisiae-derived URA3 genes [9]. A new attractive element is the A. adeninivorans-derived TRP1 gene [20]. Obviously use of such complementation markers is restricted to the existing range of respective auxotrophic hosts.
A range of ARS and CEN sequences from various sources provide elements for copy number increase.

For expression control of the heterologous gene, a TEF1 promoter like that derived from A. adeninivorans [21] or A. gossypii can be employed when addressing a large number of platforms. Other control elements are likely to elicit appropriate expression levels in a restricted number of yeasts or in a single species only: promoters of the nitrate assimilation pathway from various sources could by employed for nitrate-assimilating species, promoters of the methanol utilization pathway for methylotrophs.

The basic design of a wide-range yeast vector with a selection of components is provided in Figure 2 and Tab. 1. Two variants of a vector backbone exist, derivatives of standard E. coli plasmids, that are discriminated by the presence or absence of a f1(-) origin. A sequence of restriction sites within a MCS (multiple cloning site) has been engineered for uptake of different vector components that are provided as modules. For insertion ARS/CEN modules (module 1) are flanked by SacII/BcuI restriction sites, rDNA segments (module 2) by BcuI/Eco47III sites, selection marker modules (module 3) by Eco47III/SalI sites and expression cassettes (module 4) by SalI/ApaI sites. By easy exchange of modules, such a vector can be converted into a plasmid that is optimal for an individual platform, for instance by replacing an expression cassette with a “universal” TEF1 promoter by an element under control of a FMD promoter, optimal to drive heterologous gene expression in H. polymorpha. In variants of these basic vectors it is possible to linearize the plasmids in a way that leaves behind all bacterial DNA sequences (Figure 2 and Table 1).
 

Application of the wide-range vector to protein production in various yeasts

For proof-of-concept two combinations of modules were assessed, one applying a dominant selection marker, another one applying an A. adeninivorans-derived LEU2 gene to transformant selection in a range of respective auxotrophic host strains. In the first case GFP production was assessed in a range of yeast platforms transforming the different platform candidates in parallel with a single plasmid containing a conserved rDNA element for targeting. As reporter gene a GFP sequence was employed, which was inserted between the constitutive A. adeninivorans-derived TEF1 promoter and the S. cerevisiae-derived PHO5 terminator for expression control. The resulting plasmid was successfully used to transform A. adeninivorans, S. cerevisiae, D. hansenii, D. polymorphus, H. polymorpha and P. pastoris strains in parallel. Representatives of the various collections of transformants were tested for the recombinant protein production and all examined strains produced the GFP as shown either by Western Blot analysis or by fluorescence microscopy [9,17].

In the second example an expression/integration vector was constructed for the secretion of the pharmaceutically important interleukin-6 (IL-6), now combining an rDNA targeting sequence and an A. adeninivorans-derived LEU2 gene [19] for selection. For assessment, an expression cassette harbouring an ORF for a MF&alpha1/IL-6 fusion protein under control of the TEF1 promoter described before was used to transform leu2 auxotrophic strains of A. adeninivorans, H. polymorpha and S. cerevisiae. Representatives of the three derived strain collections efficiently secreted the recombinant cytokine. In this case, the H.polymorpha and S. cerevisiae-derived molecules were found to be of smaller size than that secreted from the A. adeninivorans host. A more detailed comparative MS analysis of tryptic peptides revealed an N-terminal truncation at position Arg8 in H. polymorpha and S. cerevisiae, but a correctly processed mature IL-6 in A. adeninivorans, probably due to the lack of a thiol protease in this dimorphic species [9,10]. This result emphasizes the need of a careful early pre-selection of a platform for the development of a production process.

Wide-range rDNA integration of multiple expression cassettes

Following a previous observation that the integrated heterologous DNA can be present as multiple units inserted in the rDNA, it was demonstrated that rDNA plasmids, each equipped with the same targeting element and the same selection marker, but bearing different reporter genes could be integrated simultaneously into the rDNA of H. polymorpha [21]. Thus, this approach provides an attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. Furthermore it is feasible to assess several yeast platforms in parallel provided the vector components follow the rationale for wide-range application described before.

This was successfully shown for the comparative assessment of different yeasts for the production of polyhydroxyalkanoates (PHA) in Debariomyces polymorphus, D. hansenii and A. adeninivorans by co-integrating and co-expressing the genes phbA, phbB and phbC of the PHA synthetic pathway from Ralstonia eutropha under control of the A. adeninivorans-derived TEF1 promoter. Following the previous examples, the vectors were further equipped with an rDNA sequence and the E. coli-derived hph gene for wide-range integration and selection. Representatives of the three resulting strain collections were found to contain all three heterologous genes as single copies mitotically stable integrated into the genome and all recombinant yeasts were able to convert efficiently the substrates acetyl-CoA and propionyl-CoA to PHA [9,23].

In yet another example the co-integration approach was applied to the improvement of IFN-&upsih secretion. IFN-&upsih was found to poorly secreted as overglycosylated proteins from various hosts³. For potential improvements strains were generated in which the gene for the cytokine was co-integrated and co-expressed together with candidate genes that could potentially influence and improve secretion and glycosylation. Of several candidate genes tried, the H. polymorpha-derived CNE1 gene encoding calnexin was found to improve secretion of the cytokine considerably. The size of the secreted product corresponded to that of core-glycosylated molecules [9]. Similar observations were made when co-expressing the S. cerevisiae-derived CMK2 gene encoding a calmodulin-dependent kinase (unpublished observations).
 

Conclusions and perspectives

Integration of heterologous DNA into a range of yeast hosts in parallel has been successfully demonstrated for transformation with a single plasmid and for co-transformation with several plasmids. The existing catalogue of elements will be constantly supplemented by new promoter elements and a range of selection markers and the respective auxotrophic hosts. A module library of genes of the synthetic and secretory pathway is under development. The CoMed vector system is very versatile and easy to handle. This important tool makes possible the simultaneous assessment of a wide range of yeast for a particular product- and process development, especially for pharmaceuticals. Having it at hand a high probability of success for an anticipated development can be envisaged.

Acknowledgement

Most of the application examples of the CoMed system described in this article were established by Dr. Kunze (IPK Gatersleben) and his group and extracted from mutual publications. His sup port in establishing the CoMed system and providing data and materials for a patent application are gratefully acknowledged.

Autoren:
Armin Merckelbach, Grit Hehmann, Georg Melmer and Gerd Gellissen

VI.. References
[1] Sudbery P.E.: Curr Opin Biotechnol 7, 517 (1996)
[2] Gellissen G.: Appl Microbiol Biotechnol. 54, 741 (2000)
[3] Gellissen G.: Hansenula polymorpha - Biology and Applications. Weinheim, Germany: Wiley-VCH (2002)
[4] Jigami Y., Odani T.: Biochim Biophys Acta 1426, 335 (1999)
[5] Bretthauer R.K. et al.: Biotechnol Appl Biochem;30:193 (1999)
[6] Guengerich L. et al.: Kück U, editor. In: The Mycota II - Genetics and Biotechnology.Heidelberg, Germany: Springer Verlag;2004, pp. 273-287.
[7] Wartmann T. et al.: Yeast 19, 849 (2002)
[8] Weydemann U et al.: Appl Microbiol Biotechnol 44, 377 (1995)
[9] Steinborn G. et al.: Microbial Cell Factories 5, 33 (2006)
http://www.microbialcellfactories.com/content/5/1/33
[10] Boeer E. et al.: FEMS Yeast Res 7, 1181 (2007)
[11] Romanos M.A. et al.: Yeast 8, 423 (1992)
[12] Kang H.A., Gellissen G.: Hansenula polymorpha. Gellissen G, editor. In: Production of recombinant proteins – Novel microbial and eukaryotic expression systems. Weinheim, Germany, Wiley-VCH; p 111 (2005)
[13] Waschk D. et al.: Characteristics of the Hansenula polymorpha genome. Gellissen G, editor. In: Hansenula polymorpha - biology and applications. Weinheim, Germany: Wiley-VCH; p 95 (2002).
[14] Ramezani-Rad M. et al.: FEMS Yeast Res 4, 207 (2003)
[15] Maleszka R., Clark-Walker G.D.: Yeast, 9 (1993)
[16] Steinborn G. et al.: FEMS Yeast Res 5, 1047-1054 (2005)
[17]Wartmann T. et al. FEMS Yeast Res 2, 363 (2002)
[18] Roesel H., Kunze G.: Curr Genet 33, 157 (1998)
[19] Wartmann T. et al. FEMS Yeast Res 3, 223 (2003)
[20] Steinborn G. et al.: J Biotechnol 127, 392 (2007)
[21] Roesel H., Kunze G.: Curr Genet 28, 360 (1995)
[22] Klabunde J. et al.: Appl Microbiol Biotechnol 58, 797 (2002)
[23] Terentiev Y. et al.: Appl Microbiol Biotechnol 64, 376 (2004)

Components of the CoMed System (Selection)

Vector components

Module 1: ARS/CEN sequences
HARS1 (H.polymorpha-derived autonomously replication sequence)
ARS (S.cerevisiae)
CEN (S.cerevisiae)

Module 2: rDNA targeting sequences
NTS2-ETS-18SrDNA-ITS1 (H.polymorpha, A.adeninivorans)

Module 3: selection marker

1. dominant
TEF1 promoter (A.gossypii; A.adeninivorans) – hph (E.coli) – TEF1 terminator (hygromycin resistance)
TEF1 promoter (A.gossypii; A.adeninivorans) – kanMX (E.coli) – TEF1 terminator (gentamycin resistance)

2. complementation
URA3 (S.cerevisiae)
LEU2 and dLEU2 (S.cerevisiae, A.adeninivorans)
TRP1 (S.cerevisiae)

Module 4: expression cassettes consisting of promoter – cloning site – terminator (terminator mostly from MOX)

wide-range
TEF1 promoter (A.adeninivorans, A.gossypii)
restricted range
NR promoter (H.polymorpha)
TPS1 promoter (H.polymorpha)
H.polymorpha-specific
MOX promoter
FMD promoter

Yeast strains (selection)

Species                                                                    Auxotrophies
Arxula adeninivorans (3 strains)                         w.t., leu2           
Hansenula polymorpha (CBS4732)                     w.t., ura3; leu2
Kluyveromyces lactis                                          w.t., met1 ura3
Pichia pastoris                                                       w.t., ura3, ura3 his3
Saccharomyces cerevisiae                                w.t., ura3, ura3 leu2 trp1 lys2
Yarrowia lipolytica                                                w.t., ura3, leu2, ura3 leu2