Phytase of the yeast Arxula adeninivorans

Of a number of yeasts screened for growth on phytic acid (inositol phosphates) as a sole source of carbon and phosphate, Arxula adeninivorans showed a particularly vigorous growth. This capacity was correlated with the presence of a high activity of secreted phytase. The crude enzyme showed an optimal temperature of at least 75°C and an optimal pH of 4.5. The level of secreted enzyme far exceeded that of previously reported yeast phytases.     

Genetic transformation and biotechnological application of the yeast Arxula adeninivorans

The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 °C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 °C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 °C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression. Received: 25 February 2000 / Received revision: 8 June 2000 / Accepted: 9 June 2000     

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.     

Novel metabolic routes during the oxidation of hydroxylated aromatic acids by the yeast Arxula adeninivorans

Aim: To complete our study on tannin degradation via gallic acid by the biotechnologically interesting yeast Arxula adeninivorans as well as to characterize new degradation pathways of hydroxylated aromatic acids. Methods and Results: With glucose‐grown cells of A. adeninivorans, transformation experiments with hydroxylated derivatives of benzoic acid were carried out. The 12 metabolites were analysed and identified by high performance liquid chromatography and GC/MS. The yeast is able to transform the derivatives by oxidative and nonoxidative decarboxylation as well as by methoxylation. The products of nonoxidative decarboxylation of protocatechuate and gallic acid are substrates for further ring fission. Conclusion: Whereas other organisms use only one route of transformation, A. adeninivorans is able to carry out three different pathways (oxidative, nonoxidative decarboxylation and methoxylation) on one hydroxylated aromatic acid. The determination of the K_M‐values for protocatechuate and gallic acid in crude extracts of cells of A. adeninivorans cultivated with protocatechuate and gallic acid, respectively, suggests that the decarboxylation of protocatechuate and gallic acid may be catalysed by the same enzyme. Significance and Impact of the Study: This transformation pathway of protocatechuate and gallic acid via nonoxidative decarboxylation up to ring fission is novel and has not been described so far. This is also the first report of nonoxidative decarboxylation of gallic acid by a eukaryotic micro‐organism.     

Physiological characterization of a microbial sensor containing the yeast Arxula adeninivorans LS3

The yeast Arxula adeninivorans LS3 is a suitable organism for use as part of a microbial sensor. In combination with an amperometric oxygen electrode the sensor offered a possibility for the physiological characterization of this yeast. About 300-400 measurements could be carried out with a single Arxula sensor. The microbial sensor was remarkably stable for over 35 days, when kept at 37 °C during the operation time and at room temperature overnight. The physiological characteristics of Arxula adeninivorans LS3 obtained with the sensor technique were identical to the data obtained with the conventional techniques. However, the sensor technique makes it additionally possible to quantify the physiological data. So the substrates ribose, citric acid, glycerol, oil and benzoate produced signals lower than 10% in comparison to the glucose signal. Fructose, xylose, sucrose, maltose, gentianose, glucosamine, glutamic acid, tryptophan, butyric acid, lauryl acid and propionic acid reached 10-70%, galactose, alanine, glycine, lysine and methionine signals were similar to the glucose signal whereas acetic acid, ethyl alcohol, capron acid, capryl acid and caproic acid reached the highest signals up to 434%.     

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 alpha-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 degrees 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 alpha-D-glucopyranosides maltotriose, maltose and isomaltose. Thus the invertase primarily is a ss-fructosidase and not an alpha-glucosidase as suggested by the homology to such enzymes.     

Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3

The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.     

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis

 The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar. Received: 12 June 1995/Received revision: 17 July 1995/Accepted: 26 July 1995     

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.     

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.     

Definition of culture conditions for Arxula adeninivorans,a rational basis for studying heterologous gene expression in this dimorphic yeast

The yeast Arxula adeninivorans is considered to be a promising producer of recombinant proteins. However, growth characteristics are poorly investigated and no industrial process has been established yet. Though of vital interest for strain screening and production processes, rationally defined culture conditions remain to be developed. A cultivation system was evolved based on targeted sampling and mathematical analysis of rationally designed small-scale cultivations in shake flasks. The oxygen and carbon dioxide transfer rates were analyzed as conclusive online parameters. Oxygen limitation extended cultivation and led to ethanol formation in cultures supplied with glucose. Cultures were inhibited at pH-values below 2.8. The phosphorus demand was determined as 1.55 g phosphorus per 100 g cell dry weight. Synthetic SYN6 medium with 20 g glucose l^?1 was optimized for cultivation in shake flasks by buffering at pH 6.4 with 140 mmol MES l^?1. Optimized SYN6 medium and operating conditions provided non-limited cultivations without by-product formation. A maximal specific growth rate of 0.32 h^?1 and short fermentations of 15 h were achieved. A pH optimum curve was derived from the oxygen transfer rates of differently buffered cultures, showing maximal growth between pH 2.8 and 6.5. Furthermore, it was shown that the applied medium and cultivation conditions were also suitable for non-limiting growth and product formation of a genetically modified A. adeninivorans strain expressing a heterologous phytase.     

Three New Cutinases from the Yeast Arxula adeninivorans That Are Suitable for Biotechnological Applications

The genes ACUT1, ACUT2, and ACUT3, encoding cutinases, were selected from the genomic DNA of Arxula adeninivorans LS3. The alignment of the amino acid sequences of these cutinases with those of other cutinases or cutinase-like enzymes from different fungi showed that they all had a catalytic S-D-H triad with a conserved G-Y-S-Q-G domain. All three genes were overexpressed in A. adeninivorans using the strong constitutive TEF1 promoter. Recombinant 6× His (6h)-tagged cutinase 1 protein (p) from A. adeninivorans LS3 (Acut1-6hp), Acut2-6hp, and Acut3-6hp were produced and purified by immobilized-metal ion affinity chromatography and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. All three enzymes from A. adeninivorans were active from pH 4.5 to 6.5 and from 20 to 30°C. They were shown to be unstable under optimal reaction conditions but could be stabilized using organic solvents, such as polyethylene glycol 200 (PEG 200), isopropanol, ethanol, or acetone. PEG 200 (50%, vol/vol) was found to be the best stabilizing agent for all of the cutinases, and acetone greatly increased the half-life and enzyme activity (up to 300% for Acut3-6hp). The substrate spectra for Acut1-6hp, Acut2-6hp, and Acut3-6hp were quite similar, with the highest activity being for short-chain fatty acid esters of p-nitrophenol and glycerol. Additionally, they were found to have polycaprolactone degradation activity and cutinolytic activity against cutin from apple peel. The activity was compared with that of the 6× His-tagged cutinase from Fusarium solani f. sp. pisi (FsCut-6hp), also expressed in A. adeninivorans, as a positive control. A fed-batch cultivation of the best Acut2-6hp-producing strain, A. adeninivorans G1212/YRC102-ACUT2-6H, was performed and showed that very high activities of 1,064 U ml⁻¹ could be achieved even with a nonoptimized cultivation procedure.     

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.     

New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison

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.