Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts
© Steinborn et al; licensee BioMed Central Ltd. 2006
Received: 21 September 2006
Accepted: 14 November 2006
Published: 14 November 2006
Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed™) system based on A. adeninivorans- and H. polymorpha- derived elements that can be introduced in a modular way.
The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli- derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFα1/IL-6 fusion was performed in a faithful way.
rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired.
The exploitation of recombinant DNA technology to engineer expression systems for heterologous protein production provided a major task during the last decades. Production procedures 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 [1, 2]. In this respect, yeasts offer considerable advantages over alternative microbial and eukaryotic cellular systems in providing low-cost screening and production systems for authentically processed and modified compounds. These organisms furthermore meet safety prerequisites in that they do not harbour pyrogens, pathogens or viral inclusions.
The initial yeast system developed for heterologous gene expression was based on the baker's yeast Saccharomyces cerevisiae. This platform has been successfully applied to the production of various FDA-approved pharmaceuticals including insulin  and HBsAg . However, when using this system, certain limitations and drawbacks are often encountered, since 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. Other restrictions are the consequence of the limited variety of carbon sources that can be utilised by this species, which limits the fermentation design options. Sometimes, the preferential use of episomal vectors leads to instabilities of recombinant strains; as a result, batch inconsistencies of production runs can be of major concern .
Therefore, alternative yeast systems have been defined that can potentially overcome the described limitations of the traditional baker's yeast. Examples include the two methylotrophic yeast species Hansenula polymorpha [5, 6] and Pichia pastoris [5, 7], the dimorphic organism Arxula adeninivorans [8, 9] and others. These organisms share the capability to utilize a broad range of carbon sources, two of them (H. polymorpha and A. adeninivorans) can assimilate nitrate and are thermotolerant species, with the latter exhibiting a temperature-dependent dimorphism with hyphae formed at elevated temperature and alterations in the extent of O glycosylation. For all systems, a range of host strains and relevant genetic elements are available.
In case of the two methylotrophic species, engineered strains have been developed that exhibit human-like N-glycosylation patterns [6, 7, 10, 11]. The genome of both species has been completely deciphered [6, 7, 12], and for H. polymorpha a microarray chip is available . In case of dimorphic A. adeninivorans species, data on chromosomes and on a partial characterization of the genomes are already available . A more detailed description of some of the platforms can be found in  and in a recent book on production of recombinant proteins and various chapters therein [1, 2, 6, 7, 9, 15].
Despite the superior characteristics of yeast hosts in various developments, there is clearly no single system that is optimal for production of all possible proteins. Predictions of a successful development for a given protein can only be made to a certain extent when restricting the initial strain engineering to a single species. The availability of a wide-range yeast vector system enables the assessment of several yeasts in parallel for their capability to produce a particular protein in desired amounts and quality [16–18]. Examples for the application of such vectors are provided in the following overview.
Results and discussion
1. Design and optimization of the CoMed™ vector system
Since vector systems of different yeast species are based on different basic vectors it is very difficult to exchange single cassettes between the yeast systems. To reduce this disadvantage the CoMed™ vector system was established containing the pCoMed™ basic vector for integration of ARS, selection markers, rDNA sequences and expression cassettes. For this purpose, the single modules are flanked by identical restriction sites and are integrated in the same location of the basic vector. In this system, various modules can be integrated. A selection of rDNA elements derived from the A. adeninivorans or H. polymorpha clusters has been assessed for suitability as targeting sequences. Particular elements of both clusters derived from an ETS-18S – 5.8S segment were found to be optimal. Due to high conservation of the included coding regions targeting of all yeast species is feasible . If for instance the combination of rDNA and the ALEU2 gene is chosen, a range of yeasts with this auxotrophy can be targeted. The same holds for the insertion of a dominant selection marker like the E.coli-derived hph gene conferring resistance to hygromycin B in all yeast species tested so far. The expression cassette is inserted in a final step as fragments derived from pre-constructed plasmids. A range of such cassette elements exists harbouring a promoter of choice, among others the A. adeninivorans- derived TEF1 promoter mentioned before, and a S. cerevisiae PHO5 terminator separated by a multiple cloning site. Again, this promoter was found to be functional in all yeast species tested so far [20, 21]. A selection of ARS sequences is available that will result in either episomal (S. cerevisiae) or chromosomally integrated plasmids (Hansenula polymorpha). However inclusion of such a sequence may reduce the range of addressible hosts.
ARS, rDNA regions, selection markers and promoter elements of the CoMed™ vector system.
■ 2 μm DNA
■ 25S rDNA
■ 18S rDNA
expression cassette (promoter)
■ FMD promoter
■ MOX promoter
■ TPS1 promoter
■ AOX1 promoter
■ TEF1 promoter
■ AHSB4m promoter
■ GAA promoter
■ ALIP promoter
■ AINV promoter
■ AXDH promoter
■ RPS7 promoter
2. Wide-range application of the CoMed™ vector system
In a first set of examples, we present the generation of recombinants based on a range of yeast species applying a single vector to transformation. In a first construct, a combination of elements was used that is suited to transform all yeast species tested so far, an rDNA-sequence for wide-range targeting and the E.coli-derived hph gene as dominant selection marker.
3. Wide-range co-integration of different plasmids
It is possible to co-integrate multiple plasmids carrying expressible heterologous genes in the ribosomal DNA by a single transformation step [20, 43]. Again, using appropriate selection markers and a functional wide-range promoter for expression control, a comparative single-step integration in a range of selected yeast platforms becomes feasible. In the following section, three promising examples are described.
3.1 Simultaneous introduction of a new metabolic pathway into different yeasts
3.2 Construction of an estrogen sensor based on recombinant A. adeninivorans cells
3.3 Assessment of secretory pathway genes for the production of IFNγ
IFNγ is produced by CD4 and CD8-positive T and NK (natural killer) cells. The mature protein consists of 146 amino acids. IFNγ is a potent anti-viral and anti-parasitic agent. It has been assessed for treatment of opportunistic infections in AIDS patients, for treatment of eosinophilia in severe atopic dermatitis and for treatment of osteopetrosis [46, 47]. Two active forms of 25 and 20 kDa exist, differing in the extent of glycosylation. Glycosylation is not required for biological activity  but for proteolytic stability . It is therefore desirable to have access to an efficient expression platform in which a cytokine can be produced as glycosylated protein. The protein has been produced in E. coli [50, 51] and in mammalian cells .
The CoMed™ vector system is a versatile system built up in a modular way. Modules comprise of ARS sequences, rDNA targeting sequences and dominant or complementation selection markers. For expression cassettes, a choice of promoters from various sources is available, separated in the module by a MCS from an S. cerevisiae-derived PHO5 terminator.
A combination of an rDNA integration sequence and a suitable selection marker enables transformation of host strains derived from a wide range of yeast species. The vectors can easily be transformed into traditional species-specific vectors.
A single type of plasmid or multiple types of plasmids can simultaneously be integrated into the genome of the various hosts.
The vector system thus provides a powerful tool to transform several yeasts in parallel at an early stage of a particular process development thereby avoiding potential cost- and time-consuming failures.
Materials and methods
Strains and media
E. coli TOP 10 [F', mcrA, Δ(mrr-hsdRMS-mcrBC), Φ80 ΔlacZ-ΔDM15, nupG, ΔlacX74, deoR, recA1, araD139, Δ(ara,leu), 7697, galU, galK, λ-, rpsL, endA1] from Invitrogen, USA, served as host strain for bacterial transformation and plasmid isolation. Strain was grown in LB medium supplemented with ampicillin (50 μg ml-1; AppliChem, Germany) when required for selection.
The yeast strains A. adeninivorans LS3 , A. adeninivorans 135 , A. adeninivorans G1211 ([aleu2 – ), D. hansenii H158 (provided by the strain collection of UFZ, Leipzig/Germany), D. polymorphus H120 (provided by the strain collection of UFZ, Leipzig/Germany), P. pastoris GS115 (his4 – Invitrogen/USA) and the H. polymorpha MedHp1 (odc1 – ) as well as S. cerevisiae C13ABYS86 (MATα leu2 ura3 his pra1 prb1 prc1 cps – ) were used as hosts. All strains were grown either under non-selective conditions in complex medium (YEPD) or under selective conditions in a yeast minimal medium (YMM) supplemented with 2% of a selected carbon source [62, 63]. Cultivation was performed at 30°C.
Agar plates were prepared by adding 1.6% (w/v) agar to the media. Hygromycin B (Roche Diagnostics, Germany) was added as 150 – 400 μg ml-1 when required for selection.
A.adeninivorans LS3, A.adeninivorans 135, A. adeninivorans G1211, D.hansenii H158, D.polymorphus H120, H. polymorpha MedHp1, P. pastoris GS115 and S. cerevisiae C13ABYS86 were transformed according to [25, 64]. Stable transformants were obtained after a sequence of passages on selective and non-selective media. After transformation of plasmids with the hph selection marker, hygromycin B-resistant colonies were selected on YEPD agar plates supplemented with 150 – 400 mg l-1 hygromycin B (200 mg l-1 for A.adeninivorans LS3 and 135, 250 mg l-1 for D.hansenii H158 and D.polymorphus H120, 400 mg l-1 for H. polymorpha MedHp1, 150 mg l-1 for P. pastoris GS115and S. cerevisiae C13ABYS86). Single colonies were isolated and grown on YEPD medium and hygromycin B at 30°C for 2 days. This step was repeated three times before the cells were plated on non-selective YEPD agar and grown for 3–5 days at 30°C. A single colony from each transformant was isolated and defined as a strain.
In case of auxothrophy complementation the transformants were selected on YMM agar plates lacking the respective amino acid.
Isolation and characterisation of nucleic acids
SDS-PAGE with 6 μg cell extract protein and Western blots were performed as described by . The dye binding method of Bradford  was used for quantitative determination of protein concentration in cell extract with bovine serum albumin as a standard protein. IL-6 and IFNγ were immunologically detected by Western blot analysis using specific anti-IL-6 or IFNγ antibodies (R&D systems/USA) for detection. Blots were stained with Western Blue Stabilized Substrate (Promega, Germany).
IFNγ samples were digested with PNGaseF (Boehringer, Mannheim) following the instructions of the supplier. Untreated and digested samples were separated and visualized as described before.
GFP expression was visualized in yeast cells by fluorescence microscopy (Axioskop, Zeiss Jena, Germany; excitation at 470 nm and detection using the BP500–530 nm emission filter). These conditions allowed visualization of GFP-mediated fluorescence avoiding a significant auto-fluorescence background. Single images (512 × 512 pixels) were collected using line-averaging eight times, each for 1 s.
Measurement of yeast dry mass and PHA
Estrogenic activity assay
A. adeninivorans G1211 transformed with the plasmids pAL-HPH-hERα and pAL-ALEU2m-GAA(xERE)-phyK (G1211/pAL-HPH-hERα – pAL-ALEU2m-GAA(xERE)-phyK) was the bio component of the assay. These cells were cultured in YMM with 2% glucose at 30°C for 48 h and stored as 50 μl aliquots. For detection of estrogenic activity, 950 μl of the samples were supplemented with 2% maltose (final concentration) and 50 μl of stationary yeast cell suspension (final OD600 nm = 1) was added. The suspension was cultured for 30 h at 30°C on a shaker.
Subsequently all samples were centrifuged for 10 min at 5000 × g to separate the cells from the cultivation medium. The extracellularly accumulated phytase activity was assessed through a modified method initially described by [70, 71]. Aliquots (25 μl) of the supernatant samples were incubated with 25 μl 0.1 M sodium-citrate (pH 3.9) containing 3.5 mM 4-nitrophenyl phosphate for 60 min at 37°C in microtitre plates. The reaction was stopped by adding 50 μl 15% TCA. After adding of 100 μl 1 N sodium hydroxide, 4-nitrophenol was measured at 405 nm with the reader "Sunrise" from Tecan (USA) .
is a trade name (abbreviation of Construction set PharmedArtis).
The authors thank R. Franz, R. Losse, I. Schmeling and H. Bohlmann for excellent technical assistance. The research work was supported by grants from the Ministry of Science and Research (Grant No. 02WU 0168), the Ministry of Economic, Nordrhein-Westfalen (TPW-9910v08), the Deutsche Bundesstiftung Umwelt (AZ 13048) and by Funds of Chemical Industry (GK).
Parts of the results presented here have been communicated at the 4th Recombinant Protein Production Meeting (Barcelona, 2006), a detailed description of the impact of CNE1 and other secretory pathway genes has been provided at the conference for 30th anniversary of the Korean Society for Microbiology, Muju, 2003 and at the 11th ICY, Rio de Janeiro, August 2004.
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