Yeast Yeast 2016; 33: 535–547 Published online 25 August 2016 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.3180 Yeast Primer Blastobotrys (Arxula) adeninivorans: a promising alternative yeast for biotechnology and basic research Anna Malak1, Kim Baronian2 and Gotthard Kunze1* 1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany School of Biological Sciences, University of Canterbury, Christchurch, New Zealand 2 *Correspondence to: G. Kunze, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466, Gatersleben, Germany. E-mail: kunzeg@ipk-gatersleben. de Received: 29 April 2016 Accepted: 24 June 2016 Abstract Blastobotrys adeninivorans (syn. Arxula adeninivorans) is a non-conventional, non-pathogenic, imperfect, haploid yeast, belonging to the subphylum Saccharomycotina, which has to date received comparatively little attention from researchers. It possesses unusual properties such as thermo- and osmotolerance, and a broad substrate spectrum. Depending on the cultivation temperature B. (A.) adeninivorans exhibits different morphological forms and various post-translational modifications and protein expression properties that are strongly correlated with the morphology. The genome has been completely sequenced and, in addition, there is a well-developed transformation/expression platform, which makes rapid, simple gene manipulations possible. This yeast species is a very good host for homologous and heterologous gene expression and is also a useful gene donor. Blastobotrys (A.) adeninivorans is able to use a very wide range of substrates as carbon and/or nitrogen sources and is an interesting organism owing to the presence of many metabolic pathways, for example degradation of n-butanol, purines and tannin. In addition, its unusual properties and robustness make it a useful bio-component for whole cell biosensors. There are currently a number of products on the market produced by B. (A.) adeninivorans and further investigation may contribute further innovative solutions for current challenges that exist in the biotechnology industry. Additionally it may become a useful alternative to existing commercial yeast strains and as a model organism in research. In this review we present information relevant to the exploitation of B. (A.) adeninivorans in research and industrial settings. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: Arxula adeninivorans; biosensor; recombinant proteins; dimorphism; yeast Introduction Yeasts are eukaryotic microorganisms that play important roles in human life. The best known is Saccharomyces cerevisiae, which has been used since ancient times for the production of various foods and beverages, such as bread, beer and wine. Through the Yeast Genome Project (YGP), it was the first eukaryotic organism to have its genome sequenced, which is recorded in the Saccharomyces genome database (http://www.yeastgenome. org/). Nevertheless, S. cerevisiae can have some Copyright © 2016 John Wiley & Sons, Ltd. limitations such as hyperglycosylation, poor secretion and/or incorrect folding of some heterologous proteins as well as relatively low robustness against osmotic and temperature stress in industrial applications. Thus other yeast systems have been sought that might not have these problems. Alternative yeast strains being employed in biotechnology includes Blastobotrys adeninivorans (Arxula adeninivorans), Ogataea polymorpha (Hansenula polymorpha), Komagataella pastoris (Pichia pastoris), Kluyveromyces lactis and Yarrowia lipolytica. This article describes the 536 special properties of B. (A.) adeninivorans that make it a first-rate system for commercial applications and an increasingly important model system for basic research. A. Malak et al. The purpose of this paper is to provide information on this interesting and useful organism. An apparently ordinary yeast with some extraordinary properties History and phylogeny of B. adeninivorans Blastobotrys adeninivorans is a relatively recently discovered yeast species. The first report of this new organism was published by Middelhoven et al., 1984. The yeast was isolated from soil and named Trichosporon adeninovorans (Middelhoven et al., 1984). Six years later Gienow et al. (1990) described the second strain, LS3 (PAR-4), isolated from wood hydrolysates in Siberia. Further strains have been isolated from chopped maize herbage in the Netherlands and from humus-rich soil in South Africa (Van der Walt et al., 1990). All of these wild-type isolates were found to possess unusual properties such as nitrate assimilation and xerotolerance and the ability to use adenine, guanine, butylamine, soluble starch, melibiose, uric acid, pentylamine, putrescine, propylamine and hexylamine as a sole source of carbon, nitrogen and energy (Middelhoven et al., 1984). The species was given the name Arxula adeninivorans (Middelhoven et al., 1991) and subsequently renamed B. adeninivorans (Kurtzman and Fell, 1998). Another yeast species initially belonging to the Blastobotrys genus was subsequently discovered and is now named Blastobotrys terrestris. Blastobotrys adeninivorans and B. terrestris exhibit some differences: for example, the ability to assimilate melibiose, D-glucosamine and galactitol (Kurtzman and Fell, 1998). Both were classified into the Trichomonascus clade, which contains anamorphic genera such as Blastobotrys, Candida and Sympodiomyces, but also the ascosporic genera Trichomonascus and Stephanoascus (Kurtzman and Robnett, 2007). It was demonstrated, based on its complete genome sequence data (Kunze et al., 2014), that B. (A.) adeninivorans is phylogenetically distant from the most characterized yeast model system, S. cerevisiae. To date, more than 170 articles related to A. adeninivorans have been published. Although this yeast is fully characterized and its genome is completely sequenced, it is still not well known. Copyright © 2016 John Wiley & Sons, Ltd. Arxula adeninivorans is temperature tolerant and able to grow at up to 48 °C without previous adaptation. Depending on the cultivation temperature, LS3 wild-type strain exhibits three morphological forms. Up to 42 °C, the cells reproduce by budding; at 42 °C, the cells form pseudomycelia and above 42 °C they become mycelial (Wartmann et al., 1995, 2000; Figure 1). The occurrence of such dimorphism is quite common in the yeasts; however, the factors, which cause the changes in phenotype, are usually different in different yeast species. For instance, in Y. lipolytica, the morphology depends principally on the pH; however, carbon and nitrogen sources and temperature can also have an effect on the phenotype (Ruiz-Herrera and Sentandreu, 2002). It was observed that under low-temperature conditions this yeast grows mainly as mycelia, whereas under hightemperature conditions, it grows as budding cells (Medoff et al., 1987; Swoboda et al., 1994), which is in contrast to A. adeninivorans (<42 °C – budding cells; 42 °C – pseudomycelia; >42 °C – mycelial). The dimorphism of A. adeninivorans is reversible, with the key factor being temperature; for example, when the temperature of a mycelial culture is reduced to <42 °C the mycelia form buds to produce new yeast cells. The morphology of A. adeninivorans is thus quite easy to control; however, the exact mechanism underlying this dimorphism is not yet known. Budding cells of the LS3 strain and mycelia of the mutant strain A. adeninivorans 135 (growing as mycelia at 30 °C), and cultivated under the same conditions, exhibit various biochemical and secretory properties. Except for the differences in dry cell weight (dcw), total RNA and intracellular protein content, the biggest difference between the two strains occurs in the total amount of secreted proteins. The mass of extracellular proteins in mycelial cultures is twofold higher than in the budding cells. Hence, as reported by Wartmann et al. (2000), the two different morphological forms exhibit differences in their Yeast 2016; 33: 535–547 DOI: 10.1002/yea Arxula adeninivorans 537 Figure 1. Temperature-dependent dimorphism of A. adeninivorans LS3. Cell morphology of A. adeninivorans LS3: budding cells (a), pseudomycelia (b) and mycelia (c). The yeast cells were cultured in YEPD medium for 18 h at 30 °C, 42 °C and 45 °C, respectively. Bright-field images were obtained using CLSM (Zeiss LSM 780, with laser 633 nm) biochemical and secretory properties. However, it is not yet known if the amount of protein produced by the different morphological forms varies for different proteins (Böer et al., 2007). The levels of post-translational modifications, specifically glycosylation or phosphorylation, in A. adeninivorans are not constant but the mechanisms leading to these post-translational modifications are not yet understood. O-Glycosylation occurs preferentially in budding cells, which indicates that the type of glycosylation is linked to the morphology. The extent of glycosylation and phosphorylation regulation, however, seems to depend only on the temperature of cultivation (Wartman et al., 2002). Hyperglycosylation is also known in S. cerevisiae and its hyperglycosylation machinery is able to create N-linked protein glycosylation terminated by mannose attached via the α-1,3 bond. This mannose-type N-glycosylation induces an allergic response in humans (Gellissen Copyright © 2016 John Wiley & Sons, Ltd. et al., 2005) and study of glycosylation in other yeasts may provide useful information on this response. The type of N-glycosylation in A. adeninivorans is still not well known. Böer et al. (2007) performed a comparative assessment experiment in A. adeninivorans, and O. polymorpha and S. cerevisiae (which are commercially available as expression systems) as hosts for the production of the human interleukin-6 (IL-6). They showed that only A. adeninivorans synthesizes the protein with the correct post-translational processing, and it was also the most productive of the three yeasts. Analysis has shown that in the case of O. polymorpha and S. cerevisiae N-terminal truncations of proteins occurred (Böer et al., 2007). In 2015, Kumari et al. compared the secretion of lipase (YlLip11p) in two different hosts: A. adeninivorans and Y. lipolytica. They observed differences in productivity, pattern of glycosylation and thermostability of the enzyme. Arxula Yeast 2016; 33: 535–547 DOI: 10.1002/yea 538 adeninivorans exhibited 1.6-fold higher productivity than the other strains and with a higher level of glycosylation, and probably therefore a higher thermostability and lower specific activity than that achieved by O. polymorpha enzyme (Kumari et al., 2015). These studies illustrate the value of using non-model systems in yeast research because these results indicate that they might have real value in improving the quality of various industrial products. Another property of A. adeninivorans is its halotolerance. Cells are able to grow on media containing up to 20% NaCl, with only slight effects on transcription levels, secretion and growth being observed up to 10% NaCl (Tag et al., 1998; Yang et al., 2000). Osmotolerance is a very desirable feature both for fermentation as well as in bioremediation and biosensors. Because of the importance of fatty acids in the biofuel, chemistry and cosmetic markets, their production in yeasts has been studied intensively. The medium-chain fatty acids (6–14 carbons) are the most valuable and occur in S. cerevisiae primarily as lipids. These fatty acids are absent in oleaginous yeasts such as Y. lipolytica. Nevertheless, yeasts remain a viable alternative to chemical synthesis because of their environmentally friendly production. It is known that, in general, low temperatures increase the fatty acid concentration in yeasts. Olstorpe et al. (2014) first described the temperature-dependent fatty acid profile of C10 to C24 chain-length fatty acids in A. adeninivorans (strains CBS 8244 and CBS 7377). They found that C16:0 is the main fatty acid present in wild-type strains (19.5–36.8% total fatty acids). C17:1 fatty acids were synthesized at up to 30.6% of total fatty acids and monounsaturated fatty acids accumulated to between 38.3% and 52.3%. Interestingly, the C18:1 chain demonstrated different profiles in different A. adeninivorans wild-type strains. The first strain, A. adeninivorans CBS 8244, did not contain C18:1 (n-5), but C18:1 (n-7) was present at 7.4% at 15 °C and 9% at 30 °C incubation temperatures, while in another strain of A. adeninivorans, CBS 7377, C18:1 (n-5) was present at 4.5% at 15 °C and 12.6% at 30 °C. C18:1 (n-7) was only just detectable and the total fatty acids at low temperatures were lower compared to the total at higher temperatures. In both strains, the general trend of the profile of the fatty acids was similar, except Copyright © 2016 John Wiley & Sons, Ltd. A. Malak et al. for C16:1 (n-7), C18:1 (n-5) and C18:3 (n-3), where the trend was opposite. These strains, however, differed in the total fatty acid concentration, which is consistent with the notion of temperature-dependent fatty acid production in yeast. Additionally the double bond index (DBI) increases with increased cultivation temperature. However, the C16:C18 ratio is similar at 15 and 30 °C (Olstorpe et al., 2014). Hence there is no predictable fatty acid composition in these yeast strains. Froissard et al. (2015) analysed 16 strains belonging to the Saccharomycotina. They showed that A. adeninivorans (wild-type strain CBS 8244) contains 50.12 ± 0.43 μg fatty acid methyl ester (FEME) mg 1 dcw after 24 h cultivation in YP medium at 28 °C. This strain, however, is devoid of C18:3 fatty acids. The authors also found that the relative amount of fatty acids in Saccharomycotina and A. adeninivorans is similar to the profile of fatty acids in Y. lipolytica, with mainly C16:0, C18:0, C18:1 (n-9) and C18:2 (n-6) molecules present. Arxula adeninivorans LS3, which, although the most characterized A. adeninivorans wild-type strain, has not yet been tested for its fatty acid profile. Genome architecture and reproduction In 2014, the A. adeninivorans LS3 genome was completely sequenced. The mitochondrial genome has a size of 31 662 bp and encodes 24 tRNAs and 15 proteins. The nuclear genome has a size of 11.8 Mb. The nuclear genome comprises four chromosomes: Arad1A (1 659 397 nt), Arad1B (2 016 785 nt), Arad1C (3 827 910 nt) and Arad1D (4 300 524 nt) with regional centromeres. There are 6116 protein-encoding genes, 33 pseudogenes and 914 introns present. A single rDNA cluster, which plays an important role in molecular tools of A. adeninivorans, is located approximately 75 kb upstream of the Arad1D chromosome’s right subtelomere (Kunze et al., 2014; Rösel and Kunze, 1996; Pich and Kunze, 1992). Although A. adeninivorans is phylogenetically distant from S. cerevisiae, it nevertheless follows the bacterial sparing rules and reads Leu CUN and Arg CGN codons, as in baker’s yeast (Kunze et al., 2014). In most cases, this is an advantage for heterologous Yeast 2016; 33: 535–547 DOI: 10.1002/yea Arxula adeninivorans gene expression. Based on the information obtained from whole genome sequence analysis of A. adeninivorans, a microarray for gene expression analysis containing 56 312 specific 539 oligonucleotides was constructed (Figure 2). This is an additional approach to investigate the changes in transcription profile that are dependent on environment conditions. Figure 2. Microarray design and hybridization for gene expression analyses in A. adeninivorans LS3. Based on 6025 annotated chromosomal sequences and 36 putative mitochondrial genes, oligos were designed using Agilent Technologies eArray software (https://earray.chem.agilent.com; design number 035454). Depending on the length of the genes up to 10 60-mers per gene were created, resulting in a total of 56 312 A. adeninivorans specific oligos. The microarray was produced by Agilent Technologies in 8 × 60 k format. Arxula adeninivorans LS3 cells cultured under different conditions were harvested and total RNA was isolated. Probe labelling and microarray hybridization (duplicates) were executed according to the manufacturer’s instructions (Agilent Technologies ‘One-Color Microarray-Based Gene Expression Analysis’, v6.5). Analysis of microarray data was performed with the R package limma (Smyth, 2005). Raw expression values were background corrected using ‘normexp’ and normalized between arrays using ‘quantile’. Differentially expressed genes were detected by fitting a linear model to log2-transformed data by an empirical Bayes method (Smyth, 2004). The Bonferroni method was used to correct for multiple testing Copyright © 2016 John Wiley & Sons, Ltd. Yeast 2016; 33: 535–547 DOI: 10.1002/yea 540 Sexual reproduction in A. adeninivorans has not been reported; thus it is considered to be a haploid asexual yeast. However, complete sequencing of the genome by Kunze et al. (2014) revealed the present of the MAT locus on chromosome Arad1D. The authors discussed the possibility that A. adeninivorans really is an asexual organism, or alternatively, that it is still able to reproduce sexually but the opposite mating strain has not yet been discovered. It may also be that the ploidy of a strain is relevant and that haploid organisms adapt and evolve faster than diploid organisms, and therefore have become the dominant form (Orr and Otto, 1994; Gerstein et al., 2011). There is also the possibility that during the adaptation process of A. adeninivorans to harsh environments this yeast became a haploid organism and lost the ability to reproduce sexually. Regardless of which factors led to the haploidy of A. adeninivorans, this property permits easy genetic manipulation (e.g. simple UV or nitrosoguanidine mutagenesis, followed by mutant selection), rapid selection of strains with desired properties, higher stability of the phenotype, rapid growth and the ability to disperse in a short period of time. On the other hand, asexual reproduction reduces molecular diversity. Samsonova et al. (1996) investigated the possibility of experimentally developing a parasexual cycle in A. adeninivorans. This experiment succeeded and most of the fusion hybrids obtained were relatively stable heterozygotes. Spontaneous mitotic segregation, which results in the misdistribution of the chromosomes and crossing-over or gene conversion, was found to be at a frequency of 0.1–1%. Molecular tools for A. adeninivorans The first successful transformation of A. adeninivorans was performed in 1998 by Rösel and Kunze. Homologous recombination of linearized cassettes containing a dominant selective marker were constructed and integrated into 25S rDNA of A. adeninivorans by gap repair. This strategy was based on previous studies on the characterization of the 25S rDNA from A. adeninivorans LS3. This rDNA sequence is similar to corresponding sequences from Candida albicans (91.7% similar), S. cerevisiae (90.5% similar) and Schizosaccharomyces pombe (83.8% Copyright © 2016 John Wiley & Sons, Ltd. A. Malak et al. similar). In most cases, one to three copies of the cassettes were detected in transformed strains. The transformants were stable and resistant to up to 2000–5000 μg ml 1 of hygromycin B. In contrast, transformation with circular plasmids was not successful. Transformation did not appear to cause changes in the morphology of the cells (Rösel and Kunze, 1998). In 2004, Terentiev et al. developed an A. adeninivorans transformation/expression platform called Xplor1. It involved a hybrid plasmid, based on E. coli and A. adeninivorans fragments. The vector contained the conserved A. adeninivorans 25S rDNA derived sequences allowing specific insertion into the host genome and the E. coli hph gene, which confers resistance. The TEF1 promoter sequence from A. adeninivorans, described by Rösel and Kunze (1995), was chosen for the expression control, while PHO5 terminator from S. cerevisiae was selected for transcription termination. These improved the efficiency of integration of expression cassette in heterologous systems and were demonstrated to function in S. cerevisiae, O. polymorpha, Pichia pastoris, Debaryomyces hansenii and Debaryomyces polymorphus. The study established the versatility of the Xplor1 platform and demonstrated activity of the A. adeninivorans TEF1 promoter in heterologous yeast species (Terentiev et al., 2004). The Xplor®2 system has been developed from Xplor1 (Böer et al., 2009a; Figure 3). Improvements to the original vector were made by a number of authors and have resulted in a versatile transformation/expression platform (Wartmann et al., 1998, 2003a, 2003b; Steinborn et al., 2007a, 2007b; Böer et al., 2009a). Xplor®2 enables heterologous gene expression with two different modules: a yeast rDNA integrative expression cassette (YRC), which targets genes into rDNA clusters, and a yeast integrative expression cassette (YIC), which targets genes randomly into genomic DNA. Both modules are included in the one vector and the presence of two different restriction sites enables simple and fast selection of the selected cassette. A multicloning site (MCS) can accommodate five modules. The antibiotic selection marker for yeast transformation has been replaced by auxotrophic selection markers, which overcomes the problems associated with the presence of antibiotic resistant markers in organisms used in industrial biotechnology. Yeast 2016; 33: 535–547 DOI: 10.1002/yea Arxula adeninivorans 541 Figure 3. Principle of the transformation procedure of A. adeninivorans based on the Xplor®2 transformation/expression platform. The system is based on a bacterial vector backbone, with yeast selection markers and expression modules inserted between two 25S rDNA segments. After the construction of the plasmids in E. coli, all bacterial sequences are removed by AscI and SbfI restriction. The choice of restriction enzyme determined whether the rDNA fragment flanks the expression cassette (AscI) or not (SbfI), so that the gene of interest can be integrated into the yeast genome via homologous recombination in the rDNA as yeast rDNA integrative cassette (YRC) or via non-homologous recombination as yeast integrative cassette (YIC) Furthermore, there are modules for constitutive and inducible gene expression (Böer et al., 2009a, 2009b; Terentiev et al., 2004; Wartmann et al., 2003a, 2003b). Homologous recombination is an efficient transformation procedure that introduces linear DNA fragments into chromosomes. It was designed to reduce the size of the expression cassettes to a minimum for efficient transformation. Passaging of the transformants results in an increase in the stability of the constructs, which is essential in yeast intended for industrial use. Copyright © 2016 John Wiley & Sons, Ltd. The Xplor®2 transformation/expression platform with YRC and YIC cassettes was also trialled for homologous recombination in Schwanniomyces occidentalis, O. polymorpha and S. cerevisiae. Both systems resulted in successful transformations in these yeasts (Álvaro-Benito et al., 2013; Kumari et al., 2015). The Xplor®2 system enables easy, rapid and efficient transformation to create stable transgenic strains for the production of industrially important proteins. Yeast 2016; 33: 535–547 DOI: 10.1002/yea 542 Arxula adeninivorans and Xplor®2 as a new system for recombinant protein production Industry not only requires new molecules but it also requires them to be produced sustainably. It is also very important to reduce costs and maximize efficiency to convert a new system developed in the laboratory into a large-scale commercial production. Hence, for many purposes, there is still the need to search for alternative organisms, able to tolerate extreme environmental conditions such as high temperature fluctuations, media with high osmolarity and product accumulation to high levels. Arxula adeninivorans contains useful enzyme-encoding genes such as glucoamylase (GAA – Bui et al., 1996a), tannase (ATAN1 – Böer et al., 2009c) and cutinase (ACUT1, ACUT2, ACUT3 – Bischoff et al., 2015), which make this yeast a useful gene donor. Homologous gene expression in A. adeninivorans Many enzyme-encoding genes and their regulation have been studied in A. adeninivorans and many of its enzymes have been biochemically characterized. Most of the secreted proteins are involved in the degradation of different chemical compounds. One of the potential markets for A. adeninivorans enzymes is the food and feed industries. The extracellular invertase, encoded by AINV gene, possesses high β-fructosidase and low α-glucosidase activity (Böer et al., 2004). Invertases are used widely, especially in the confectionery industry and in the pharmaceutical industry for the production of probiotics. Another enzyme is the intracellular xylitol dehydrogenase, encoded by the AXDH gene (Böer et al., 2005a). This enzyme can be applied in the food industry for xylitol production for use as an alternative sweetener (Winkelhausen and Kuzmanova, 1998) and also for bioethanol production (Matsushika et al., 2008). In 2009, Böer et al. (2009c) characterized the ATAN1 gene encoding tannin acyl hydrolase. This enzyme is involved in the hydrolysis of tannins, which are widely distributed in plants. Although tannases are produced by several microorganisms, they have limitations for industrial applications such as the amount produced, the intracellular localization of enzyme and a limited substrate spectrum. In Copyright © 2016 John Wiley & Sons, Ltd. A. Malak et al. contrast, Atan1p has a wide substrate spectrum and 97% is secreted into the medium, which makes this enzyme easy and relatively inexpensive to purify. Currently, a transgenic A. adeninivorans strain produces tannase with an activity of 1642 U l 1 during growth in a shake flask and 51 900 U l 1 in fed-batch fermentation. Tannin acyl hydrolase is currently used in the food, feed, cosmetics and pharmaceutical industries and also in environmental bioremediation (Böer et al., 2009c, 2011; Kaiser et al., 2010). Other enzymes of industrial importance produced by A. adeninivorans are the cutinases. These enzymes can be used for the degradation of natural and synthetic polymers as well as for selective esterification of fatty acids (Suzuki et al., 2014; Masaki et al., 2005). All three cutinases have been purified and their biochemical properties determined. The activity of his-tagged cutinase 2 (Acut2p), produced by A. adeninivorans G1212/YRC102-ACUT2-6H cultivated under non-optimized fed-batch conditions, reached 1064 U ml 1 (Bischoff et al., 2015). Although cutinases are classified as very unstable enzymes, PEG 200 has been shown to significantly stabilize these enzymes. Activity was observed for more than 24 h and the addition of PEG 200 to the reaction mixture also improved the enzyme activity by up to 200%. Other industrially significant enzymes obtained from A. adeninivorans are glucoamylase, lipase, transaldolase and acid phosphatase (Bui et al., 1996b; Böer et al., 2005b; El Fiki et al., 2007; Kaur et al., 2007; Minocha et al., 2007). Some metabolic pathways in A. adeninivorans, have also been investigated. An example is the purine degradation pathway, comprising the enzymes adenine deaminase, xanthine oxidoreductase, urate oxidase and guanine deaminase, which has been shown to be useful in the production of food with reduced purine levels (Jankowska et al., 2013a, 2013b; Trautwein-Schult et al., 2013, 2014). A second example is the tannin degradation pathway, which has industrial importance. The exact mechanisms of this metabolic pathway have not yet been elucidated. However, early investigations have been undertaken (Sietmann et al., 2010). Arxula adeninivorans is able to use tannic acid and gallic acid (one of the hydrolysis products of tannins) as sole sources of carbon. Tannin acyl hydrolase, the enzyme involved in the first step of tannin degradation in Yeast 2016; 33: 535–547 DOI: 10.1002/yea Arxula adeninivorans A. adeninivorans, has received particular attention because of its potential use in detannification of food, feed, beverages and cosmetics (Böer et al., 2009a, 2011). Expression of heterologous genes in A. adeninivorans Arxula adeninivorans is also suitable for commercial production of recombinant protein. In 2007 Böer et al. demonstrated the production of the correctly processed form of human interleukin-6 in A. adeninivorans. The second human protein expressed in A. adeninivorans was human interferon α2a (IFNα2a). The concentration was 1 mg l 1, which is fivefold higher than the concentration produced by S. occidentalis (Álvaro-Benito et al., 2013). Other heterologous proteins produced in A. adeninivorans include lipase 11 from Y. lipolytica (YlLip11p) (Kumari et al., 2015) and β-galactosidase from Kluyveromyces lactis. This β-galactosidase is able to perform selective hydrolysis of anomeric mixtures such as allyl α-D-gal and allyl β-D-gal, where only the allyl β-D-gal anomer was hydrolysed by the enzyme, with no cross-reactivity to the α-anomer. This process can be used to replace the rather difficult synthetic process. The products obtained by the reaction carried out by this enzyme are used for the production of tensides and play a major role in medicine as precursor for the production of glycolipids and glycoproteins (Rauter et al., 2013). Additionally, the different glycosylation patterns seen in A. adeninivorans, S. cerevisiae and O. polymorpha offer the opportunity to trial different variants of a protein. The enzymatic synthesis of enantiomerically pure alcohols has recently been investigated. Because stereoisomers are often recognized as two different compounds by the biochemistry of living organisms, they frequently behave differently in cells. Ideally, only the stereoisomer with the desired activity should be included in therapeutics. Biological synthesis results in the production of only one stereoisomer whereas chemical synthesis always results in a racemic mixture. The production of stereo-selective alcohols has been demonstrated in A. adeninivorans strains. The expression of the RrADH gene encoding alcohol dehydrogenase from Rhodococcus ruber and the BmGDH gene encoding glucose dehydrogenase Copyright © 2016 John Wiley & Sons, Ltd. 543 from Bacillus megaterium enabled the synthesis of 98% pure 1-(S)-phenylethanol with simultaneous regeneration of the cofactor (Rauter et al., 2014a). Permeabilization and immobilization of A. adeninivorans cells increased the stability and reusability of the construct and established a method that can be adapted for use with other enzymes (Rauter et al., 2014b). For example, the construction of an A. adeninivorans strain to produce 1-(R)-phenylethanol resulted in the synthesis of a single isomer with no detectable by-products present (Rauter et al., 2015). In 1992, Büttner et al. (1992) described the alcoholic fermentation in a number of A. adeninivorans wild-type strains. The authors were investigating the direct conversion of starch to ethanol in aerobic and anaerobic cultivation conditions at different temperatures. The strains produced 9–17 g l 1 ethanol at 30 °C but much less at 45 °C (0.2–0.5 g 1). In addition, it was found that A. adeninivorans could use n-butanol as the sole source of carbon and energy for the organism (Kunze et al., 2014). Furthermore, it is possible to transfer new metabolic pathways into the yeast, e.g. for n-butanol synthesis. In 2012 a patent was granted for a strain of A. adeninivorans that can synthesis n-butanol (Kunze and Hähnel, 2012). These examples indicate that A. adeninivorans is very useful host for the production of recombinant proteins and other chemicals. Arxula adeninivorans as a biocomponent for biosensors Rapidly growing pharmaceutical, chemical and cosmetic markets have contributed to the improvement of human health and the quality of life. However, an unintended side effect has been the increasing contamination of surface and ground water and the production of wastewater with molecules that are difficult to detect and to remediate, such as mammalian hormones. One consequence, for example, has been a strong influence on gender determination in fish, leading to gender imbalance in some fish populations (Hunter et al., 1986). In 1998 Tag et al. reported the resistance of A. adeninivorans to high concentrations of sodium chloride in water (Tag et al., 1998) which, with the previously described properties, makes it Yeast 2016; 33: 535–547 DOI: 10.1002/yea 544 possible to construct biosensors for use in brackish wastewater and other contaminated waters. In 2006 Hahn et al. developed the first A. adeninivorans cell-based oestrogen biosensor (A-YES). Recombinant strains containing the human oestrogen receptor α (hERα) are controlled by a strong A. adeninivorans-derived TEF1 promoter and a phytase reporter gene from Kliebsiella sp. under the separate control of the A. adeninivorans-derived glucoamylase promoter (GAA), containing the oestrogen-responsive element. Oestrogen stimulated the production and export of phytase, which was detected by an enzyme assay. It allowed the specific, sensitive and reproducible detection of oestrogen in wastewater within 30 h without prior sample concentration (Hahn et al., 2006). In the next iteration of the sensor (nAES), the detection time was reduced to between 7 and 25 h, depending on the oestrogen concentration (Kaiser et al., 2010). In 2011 the ‘EstraMonitor’ was developed as the first automated biosensor system. It contained immobilized transgenic A. adeninivorans cells that were reusable and allowed semi-online measurement (Pham Thi et al., 2012). The EstraMonitor version was further improved in 2013, allowing continuous and semi-online monitoring of oestrogenic compounds in wastewater with NaCl concentrations as high as 5% (Pham Thi et al., 2013). Some yeast species, such as C. albicans and Paracoccidioides brasiliensis, possess an oestrogen binding protein (Ebp1p), which can be used to detect oestrogen. Unfortunately they are pathogenic yeasts, which is a potential risk for human health. Chelikani et al. (2012) showed that A. adeninivorans has no detectable intrinsic response to oestrogen compounds, and Vijayan et al. (2015) presented the first transgenic A. adeninivorans strain to express the EBP1 gene from C. albicans. A single-use electrode, with recombinant oestrogen binding protein for use with a portable potentiostat, can determine oestrogen concentrations in the range of observed environmental concentrations within 2 min. Sensors based on transgenic A. adeninivorans strains for detection of other molecules, such as omeprazole, lansoprazole (Pham Thi et al., 2015) and progesterone (Chamas et al., 2015a), have also been developed. Two bioassays for photometric and spectrophotometric detection of glucocorticoids, ACopyright © 2016 John Wiley & Sons, Ltd. A. Malak et al. YGS and A-YGFS, are operational but not yet commercialized (Pham Thi et al., 2016). Furthermore, an HER-2 cancer cell detector utilizing surface plasmon resonance (SPR) has been constructed for the rapid diagnosis of a particularly aggressive type of breast cancer (Chamas et al., 2015b). Commercial products At present, there are 10 commercially available products based on the yeast A. adeninivorans. The first protein produced by this non-traditional yeast was a recombinant tannase (Atan1p; Böer et al., 2011). Today it is also possible to order tannase and the three A. adeninivorans cutinases (Bischoff et al., 2015 – Acut1p, Acut2p, Acut3p) from ASA Spezialenzyme GmbH (Germany). ‘Quo data’ GmbH (Germany) has commercialized an A-YES kit for the detection of oestrogenic activity, and ‘new diagnostics’ GmbH (Germany) offers diagnostic kits for dioxin (A-YDS Kit), oestrogens in ultrapure and potable waters (A-YES®_aqua1.1), oestrogens in saline waters (A-YES®_aqua+1.1) androgens (A-YAS®) and progesterone (A-YPS_aqua). All these diagnostic kits use transgenic A. adeninivorans cells as the detection element. Conclusion Investigation of A. adeninivorans suggests a major potential for this yeast in basic research and in industrial applications. It is a non-pathogen and widespread in some environments. The ability to grow under a wide range of environmental conditions is an advantage and the high level of secreted molecules enables exploitation of the organism for the production of biochemicals. The presence of many degradative pathways also makes A. adeninivorans useful in both bioremediation and in the food industry. A well-developed transformation/expression system, the fully sequenced haploid genome makes it possible to design recombinant strains that are highly stable. Promising future applications include biofuel production, synthesis of Yeast 2016; 33: 535–547 DOI: 10.1002/yea Arxula adeninivorans enantiomerically pure biochemicals, and construction of new cell biosensors. 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