Yeasts combine the 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. Yeasts thus provide attractive platforms for the production of recombinant proteins. Here, four important species are presented and compared: the methylotrophic Hansenula polymorpha and Pichia pastoris, distinguished by an increasingly large track record as industrial platforms, and the dimorphic species Arxula adeninivorans and Yarrrowia lipolytica, not yet established as industrial platforms, but demonstrating promising technological potential, as discussed in this article.

A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.

In the present paper, we based a search for candidates underlying different levels of salinity tolerance during germination in the Oregon Wolfe Barley mapping population (DOM ¥ REC) by proteomic profiling of the mature grain of lines showing differing levels of salinity tolerance. By contrasting the parents DOM and REC, displaying divergent stress responses, and two tolerant and two sensitive segregants, six protein spots were identified that showed a differential abundance between the tolerant and the sensitive lines. The tolerant lines expressed a higher level of 6-phosphogluconate dehydrogenase and glucose/ribitol dehydrogenase (Glc/RibDH). Both proteins were heterologously over-expressed in an osmo-sensitive yeast strain and over-expression of Glc/RibDH resulted in an enhanced ability of yeast transformants to grow on salt containing media. A quantitative trait locus (QTL) analysis of the population germinating at different salt concentrations led to the identification of two chromosome regions on 5H and one on 7H associated with salt stress response. A dense barley transcript map was employed to map the genomic region of all identified proteins. Two of these, heat-shock protein 70 and Glc/RibDH, co-localized with the identified QTL on chromosome 5H. The putative functional role of the candidates is discussed.

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 wildtype 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.

A transformation system has been developed for the dimorphic yeast Arxula adeninivorans based on a stable integration of the donor DNA into ribosomal DNA. For this purpose a cassette was constructed which contains the E. coli hph gene, conferring hygromycin B resistance, fused to the 5' expression signals of the A. adeninivorans TEF1 gene, encoding the translation elongation factor EF-1alpha, and the transcription termination region of the Saccharomyces cerevisiae PHO5 gene. This cassette was fused into the 25S rDNA of A. adeninivorans. Linearization of this vector was required for high transformation frequencies. The vector was integrated in multiple copies into the 25S rDNA by homologous recombination. Copy number was not altered even after the growth of transformants for 15 generations under non-selective growth conditions. Microscopical analyses revealed that integration of the transformed plasmid did not influence the dimorphism, which is triggered at 42 degrees C for both transformed and non-transformed cells.

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.

BackgroundThe industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant.ResultsThe sequencing of strain LS3 revealed that the nuclear genome of A. adeninivorans is 11.8 Mb long and consists of four chromosomes with regional centromeres. Its closest sequenced relative is Yarrowia lipolytica, although mean conservation of orthologs is low. With 914 introns within 6116 genes, A. adeninivorans is one of the most intron-rich hemiascomycetes sequenced to date. Several large species-specific families appear to result from multiple rounds of segmental duplications of tandem gene arrays, a novel mechanism not yet described in yeasts. An analysis of the genome and its transcriptome revealed enzymes with biotechnological potential, such as two extracellular tannases (Atan1p and Atan2p) of the tannic-acid catabolic route, and a new pathway for the assimilation of n-butanol via butyric aldehyde and butyric acid.ConclusionsThe high-quality genome of this species that diverged early in Saccharomycotina will allow further fundamental studies on comparative genomics, evolution and phylogenetics. Protein components of different pathways for carbon and nitrogen source utilization were identified, which so far has remained unexplored in yeast, offering clues for further biotechnological developments. In the course of identifying alternative microorganisms for biotechnological interest, A. adeninivorans has already proved its strengthened competitiveness as a promising cell factory for many more applications.

SummaryBackground Usage of hair dye products containing p-phenylenediamine (PPD) is a concern for PPD-allergic individuals. Objectives The present study investigates the role of dose and exposure time on elicitation of allergic contact dermatitis under conditions of permanent hair dyeing. Methods Elicitation responses after application of a typical hair dye product containing 2% PPD for 30 min followed by rinsing were analysed in 38 PPD-allergic individuals with a documented history of hair dye-related allergy. Skin binding experiments in vitro were performed to distinguish the dose available for elicitation from the dose applied. Results A positive reaction was elicited in 20 of 20 patients with grades ++ to +++ and 12 of 18 with grade + according to the classification of the International Contact Dermatitis Research Group. Under conditions of diagnostic patch testing (48 h exposure), the dose available for elicitation is more than 10-fold higher compared with the dose available for hair dyeing (30-min exposure, rinsing of product). Conclusions This investigation demonstrates that under simulated hair dye use conditions the actual exposure to PPD is more than an order of magnitude lower than under diagnostic patch testing, although sufficient to elicit a clearly noticeable reaction in 84% of PPD patch test-positive individuals.