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.

A novel vector element providing multicopy vector integration in Arxula adeninivorans.

An Arxula adeninivorans vector element has been identified that provides multicopy integration in an atrp1 host strain. The element consists of the ATRP1 selection marker fused to a newly generated truncated ALEU2 promoter of 53 bp. In the described example eight copies of an amyA expression vector encoding heterologous alpha-amylase from Bacillus amyloliquefaciens are integrated in the genome of the recombinant strain instead of a single copy observed when using the ATRP1 element with the complete promoter. The high copy number results in strains of superior productivity for a secreted recombinant alpha-amylase. The vector design enables the integration of a small vector fragment that consists of yeast DNA only providing high transformation frequencies and a high mitotic stability.

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.

A wide-range yeast vector (CoMed) system has been applied to the comparative assessment of three different yeast platforms for the production of human interleukin-6. A vector equipped with an rRNA gene targeting sequence and an Arxula adeninivorans-derived LEU2 gene was used for simultaneous transformation of auxotrophic A. adeninivorans, Hansenula polymorpha and Saccharomyces cerevisiae strains. IL6 was expressed under control of the strong constitutive A. adeninivorans-derived TEF1 promoter, which is functional in all yeast species analyzed so far. Secreted IL-6 was found to be correctly processed from an MFalpha1-IL6 precursor in A. adeninivorans only, whereas N-terminally truncated proteins were observed in H. polymorpha and S. cerevisiae.

Construction of an Arxula adeninivorans host-vector system based on trp1 complementation.

A host/vector expression system based on an Arxula adeninivorans Delta atrp1 gene disruption mutant has been constructed. For this purpose the ATRP1 gene encoding a phosphoribosyl anthranilate isomerase was isolated from the yeast A. adeninivorans and its genome locus was characterized. The Delta atrp1 mutant was generated applying an amplified DNA fragment containing the ALEU2m gene flanked by ATRP1 gene sequences of some 750 bp. The generated auxotrophic host strain was transformed with the plasmid pAL-ATRP1-amyA, which contains the ATRP1 gene as selection marker and the 25S rDNA for targeting. For expression assessment, the plasmid was equipped with an expression cassette consisting of the Bacillus amyloliquefaciens-derived amyA gene fused to the constitutive A. adeninivorans-derived TEF1 promoter and Saccharomyces cerevisiae-derived PHO5 terminator. Transformants contained a single chromosomal copy of the heterologous DNA and were found to be mitotically stable. In initial fermentation trials on a 200 ml shake flask scale maximal alpha-amylase product levels of ca. 300 nkat ml(-1) were observed after 72 h of cultivation with more than 95% of the recombinant alpha-amylase accumulated in the culture medium.

APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity.

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36-39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60 degrees C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.

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

BACKGROUND: 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. RESULTS: 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 MFalpha1/IL-6 fusion was performed in a faithful way. CONCLUSION: 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.

Assessment of Hansenula polymorpha and Arxula adeninivorans-derived rDNA-targeting elements for the design of Arxula adeninivorans expression vectors.

Different targeting sequences derived from the Arxula adeninivorans and Hansenula polymorpha rDNA clusters were tested in A. adeninivorans integration/expression vectors. For element identification, the rDNA unit of A. adeninivorans (accession number ) was first isolated and characterized in addition to the known H. polymorpha unit. The rDNA is a cluster of some forty 7653-bp units without the 5S rDNA gene. The selected elements were integrated into a set of A. adeninivorans expression/integration vectors harbouring a TEF1 promoter - amyA ORF - PHO5 terminator sequence as reporter gene. No differences in mitotic stability, copy number and transformation frequency were observed. All transformants harboured a single copy integrated into the rDNA by a homologous recombination. In contrast, the choice of the rDNA targeting sequence was found to be of impact on productivity. Use of ETS-18S-5.8S fragments from both organisms resulted in a more than 50% increase in comparison to the use of other elements, independent of the orientation within the vector.

bac genes for recombinant bacilysin and anticapsin production in Bacillus host strains.

The genes encoding the biosynthesis of the dipeptide bacilysin and its antibiotic constituent anticapsin were isolated from several strains of Bacillus subtilis as well as B. amyloliquefaciens and B. pumilus. The ywfBCDEF genes of B. subtilis 168 were shown to carry the biosynthetic core functions and were renamed bacABCDE. Mutation of the bacD gene or transformation of the bacABC genes into a B. subtilis Delta (ywfA-bacABCDE) deletion mutant led to the accumulation of anticapsin, which was fourfold higher after transformation of the bacABC genes into a bacD mutant. The genes bacD and bacE proved to encode the functions of amino acid ligation and self-protection to bacilysin, respectively. Amplification of the bacABCDE gene cluster in a bacAB gene-deficient host strain of B. amyloliquefaciens resulted in a tenfold bacilysin overproduction. Some host strains required distinct glucosamine and yeast extract supplements in order to prevent suicidal effects of the recombinant antibiotic production. The bac genes from different Bacillus species revealed the same arrangement and 72.6-88.6% of sequence identity.