Erik Böer

Yeast expression platforms

Yeasts provide attractive expression platforms. They combine ease of genetic manipulations and the option for a simple fermentation design of a microbial organism with the capabilities of an eukaryotic organism to secrete and to modify a protein according to a general eukaryotic scheme. For platform applications, a range of yeast species has been developed during the last decades. We present in the following review a selection of established and newly defined expression systems. The review is concluded by the description of a wide-range vector system that allows the assessment of the selected organisms in parallel for criteria like secretion or appropriate processing and modification in a given case.

Atan1p—an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme

The tannase‐encoding Arxula adeninivorans gene ATAN1 was isolated from genomic DNA by PCR, using as primers oligonucleotide sequences derived from peptides obtained after tryptic digestion of the purified tannase protein. The gene harbours an ORF of 1764 bp, encoding a 587‐amino acid protein, preceded by an N‐terminal secretion sequence comprising 28 residues. The deduced amino acid sequence was similar to those of tannases from Aspergillus oryzae (50% identity), A. niger (48%) and putative tannases from A. fumigatus (52%) and A. nidulans (50%). The sequence contains the consensus pentapeptide motif (–Gly–X–Ser–X–Gly–) which forms part of the catalytic centre of serine hydrolases. Expression of ATAN1 is regulated by the carbon source. Supplementation with tannic acid or gallic acid leads to induction of ATAN1, and accumulation of the native tannase enzyme in the medium. The enzymes recovered from both wild‐type and recombinant strains were essentially indistinguishable. A molecular mass of ∼320 kDa was determined, indicating that the native, glycosylated tannase consists of four identical subunits. The enzyme has a temperature optimum at 35–40 °C and a pH optimum at ∼6.0. The enzyme is able to remove gallic acid from both condensed and hydrolysable tannins. The wild‐type strain LS3 secreted amounts of tannase equivalent to 100 U/l under inducing conditions, while the transformant strain, which overexpresses the ATAN1 gene from the strong, constitutively active A. adeninivorans TEF1 promoter, produced levels of up to 400 U/l when grown in glucose medium in shake flasks. Copyright

High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30 degrees C and mycelia at >42 degrees C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30 degrees C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.

The ALEU2 gene--a new component for an Arxula adeninivorans-based expression platform.

The ALEU2 gene, encoding beta-isopropylmalate dehydrogenase, was isolated from the non-conventional yeast Arxula adeninivorans. The isolated gene harbours an open reading frame of 1086 bp, encoding a putative protein of 362 amino acids. The derived protein sequence shares a high degree of homology with other fungal beta-isopropylmalate dehydrogenases thus confirming the identity of the gene. The isolated ALEU2 gene was tested for its suitability to complement the auxotrophy of an A. adeninivorans aleu2 host. For this purpose the plasmid pAL-ALEU2m which contains the ALEU2 gene as a selection marker and the 25S rDNA for targeting was employed in transformation experiments. Transformants harboured a single copy of the heterologous DNA and were found to be mitotically stable. For assessment of heterologous gene expression, two model genes were incorporated into the vector: the GFP gene, encoding intracellular green fluorescent protein, and the HSA gene, encoding the secreted human serum albumin. For expression control, both gene sequences were fused to the constitutive A. adeninivorans-derived TEF1 promoter and the Saccharomyces cerevisiae-derived PHO5 terminator. In the respective recombinant strains the GFP was localised in the cytoplasm, whereas more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials using a 200-ml shake flask, maximal HSA product levels were observed after 96 h of cultivation.

Xplor® 2—an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans

Combining ease of genetic manipulation and fermentation with the ability to secrete and to glycosylate proteins in the basic eukaryotic manner, Arxula adeninivorans provides an attractive expression platform. Based on a redesign of the basic vector, a new Arxula vector system, Xplor® 2, for heterologous gene expression was established, which allows (1) the construction of expression plasmids for supertransformation of A. adeninivorans strains secreting target proteins of biotechnological interest and (2) the integration of small vector cassettes consisting of yeast DNA sequences only. For this purpose, a set of modules including the ATRP1m selection-marker module, expression modules for constitutive expression of the genes phyK (Klebsiella-derived phytase) and IFNα2a (human interferon α), the HARS (Hansenula polymorpha autonomous replication sequence) for autonomous replication and the chaperone module AHSB4 promoter –HpCNE1 gene (calnexin) –PHO5 terminator to improve secretion efficiency were constructed and integrated in various combinations in the basic vector Xplor® 2. After removal of the complete Escherichia coli-based plasmid parts (resistance marker, ColE1 ori and f1(−) origin), the remaining yeast-based linear vector fragment with or without rDNA targeting sequences were transformed as yeast rDNA integrative expression cassettes and yeast integrative expression cassettes (YICs), respectively, and the resulting strains were tested for their capacity to secrete PhyK or IFNα2a. Maximal expression levels were consistently obtained using YICs for transformation irrespective of whether or not they carry HARS and/or calnexin modules. It is recommended that at least 50 such transformants be analyzed to ensure selection of the best transformants.

Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts

BackgroundYeasts 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.ResultsThe 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.ConclusionrDNA 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.

An extracellular lipase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ALIP1 gene and characterization of the purified recombinant enzyme

The lipase‐encoding Arxula adeninivorans ALIP1 gene was isolated using fragments of lipase isolates obtained by trypsin digestion for the definition of oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1347 bp encoding a 420 amino acid protein of some 50 kDa preceded by an N‐terminal 28 prepro‐secretion sequence. The deduced amino acid sequence was found to be similar to the lipases from Candida albicans and C. parapsilosis (34–38% identity) and more distantly related to other lipases. The sequence contains the consensus pentapeptide motif (–Gly–X–Ser–X–Gly–) that forms a part of the interfacial lipid recognition site in lipases. The expression of the gene is regulated by carbon source. In media supplemented with Tween 20, induction of the ALIP1 gene and accumulation of the encoded lipase in the medium is observed, thus demonstrating gene regulation by lipophilic compounds. The enzyme characteristics are analysed from isolates of native strains as well as from those of recombinant strains expressing the ALIP1 gene under control of the strong A. adeninivorans‐derived TEF1 promoter. For both proteins a molecular mass of 100 kDa was determined, indicating a dimeric structure, a pH optimum at pH 7.5 and a temperature optimum at 30 °C. The enzyme hydrolyses all ester bonds in all triglyceride substrates tested. Middle‐sized chain fatty acids are more efficiently hydrolysed than short‐ and long‐chain fatty acids, with the highest activity on C8/C10 fatty acid esters pNP‐caprylate, pNP‐caprate and tricaprylin. Copyright

A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements

AbstractAn Arxula adeninivorans integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1 promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level.

Characterization of the AINV gene and the encoded invertase from the dimorphic yeast Arxula adeninivorans

The invertase-encoding of AINV gene Arxula adeninivorans was isolated and characterized. The gene includes a coding sequence of 2700 bp encoding a putative 899 amino acid protein of 101.7 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of α-glucosidases from different sources. The gene activity is regulated by carbon source. In media supplemented with sucrose induction of the AINV gene and accumulation of the encoded invertase in the medium was observed. In addition the extracellular enzyme level is influenced by the morphological status of the organism, with mycelia secreting the enzyme in titres higher than those observed in budding yeasts. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AINV gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins a molecular mass of 600 kDa was determined, a pH optimum at pH 4.5 and a temperature optimum at 55 °C. The preferred substrates for the enzyme included the ß-D-fructofuranosides sucrose, inulin and raffinose. Only a weak enzyme activity was observed for the α-D-glucopyranosides maltotriose, maltose and isomaltose. Thus the invertase primarily is a ß-fructosidase and not an α-glucosidase as suggested by the homology to such enzymes.

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

The yeast Arxula adeninivorans is characterized by a temperature‐dependent dimorphism. A. adeninivorans grows as budding cells at temperatures up to 42°C, but forms mycelia at higher temperatures. A strong correlation exists between morphological status and iron uptake, achieved by two transport systems that differ in iron affinity. In the presence of high Fe(II) concentrations (>2 µm), budding cells accumulate iron concentrations up to seven‐fold higher than those observed in mycelia, while at low Fe(II) concentrations (<2 µm), both cell types accumulate similar amounts of iron. The copper‐dependent Fe(II) oxidase Afet3p, composed of 615 amino acids, is a component of the high‐affinity iron transport system. This protein shares a high degree of homology with other yeast iron transport proteins, namely Fet3p of Saccharomyces cerevisiae, Cafet3p of Candida albicans and Pfet3p of Pichia pastoris. Expression of the AFET3 gene is found to be strongly dependent on iron concentration but independent of the morphological stage; however, cell morphology was found to influence post‐translational modifications of the gene product. O‐glycosylation was observed in budding cells only, whereas N‐glycosylation occurred in both cell types. The N‐glycosylated 103 kDa glycoprotein matures into the 108.5 kDa form, further characterized by serine phosphorylation. Both N‐glycosylation and phosphorylation occur at low iron concentrations (≤5 µm). The mature Afet3p of 108.5 kDa is uniformly distributed within the plasma membrane in cells of both morphological stages. Copyright