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.

Extracellular expression of YlLip11 with a native signal peptide from Yarrowia lipolytica MSR80 in three different yeast hosts.

Lipase YlLip11 from Yarrowia lipolytica was expressed with a signal peptide encoding sequence in Arxula adeninivorans, Saccharomyces cerevisiae and Hansenula polymorpha using the Xplor®2 transformation/expression platform and an expression module with the constitutive Arxula-derived TEF1 promoter. The YlLip11 signal peptide was functional in all of the yeast hosts with 97% of the recombinant enzyme being secreted into the culture medium. However, recombinant YlLip11 with His Tag fused at C-terminal was not active. The best recombinant YlLip11 producing A. adeninivorans G1212/YRC102-YlLip11 transformant cultivated in shake flasks produced 2654 U/L lipase, followed by S. cerevisiae SEY6210/YRC103-YlLip11 (1632U/L) and H. polymorpha RB11/YRC103-YlLip11 (1144U/L). Although the biochemical parameters of YlLip11 synthesized in different hosts were similar, their glycosylation level and thermo stability differed. The protein synthesized by the H. polymorpha transformant had the highest degree of glycosylation and with a t1/2 of 60min at 70°C, exhibited the highest thermostability.

Purification and immunodetection of the complete recombinant HER-2[neu] receptor produced in yeast.

For the first time, the full length recombinant HER-2[neu] receptor has been produced in a yeast (Arxula adeninivorans). It is one of the most studied membrane receptors in oncology and is involved in aggressive tumor formation. A yeast integration rDNA cassette containing the human gene coding for the HER-2[neu] protein was constructed and a screening procedure was performed to select the most productive transformant. Different detergents were tested for efficient solubilization of the membrane bound protein, with CHAPS giving the best results. To increase the yield of the recombinant protein from HER-2[neu] producing A. adeninivorans, optimal culture parameters were established for cultivation in bioreactor. The recombinant protein was subsequently assayed using ELISA and SPR immunoassays systems with antibodies raised against two different epitopes of the human receptor. In both cases, elution fractions containing the recombinant HER-2[neu] receptor successfully reacted with the immunoassays with limits of quantification below 100ngml(-1). These results demonstrate that the full length recombinant HER-2[neu] reported here has the potential to be a new standard for the detection of HER-2 type cancer.

Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater.

This study describes the development of a bioassay to detect the presence of progesterone and progesterone-like molecules in wastewater samples. The basis of the bioassay is the integration of the human progesterone receptor gene into the yeast Arxula adeninivorans for the constitutive synthesis of the receptor. After incubation, binding of the analyte to the receptor induces the production of a reporter protein. Two reporter proteins were compared for detection parameters such as half-maximal activity (EC50), limit of detection (LoD) and limit of quantification (LoQ). When the extracellular phytase K was used, an EC50 value of 155 ng L(-1) and a LoD of 27 ng L(-1) progesterone were obtained after 4 h incubation, while use of the fluorescent dsRED as the reporter protein, resulted in an EC50 of 320 ng L(-1) and a LoD of 65 ng L(-1) after 20 h incubation. Use of phytase K as the reporter protein offers decreased incubation time and increased sensitivity; however the dsRED reporter system is less labor-intensive. Additionally, the affinity of known agonists and antagonists of the human progesterone receptor was determined. The utility of this bioassay was confirmed by measuring total progesterone equivalent concentration of samples from a wastewater treatment plant. The A. adeninivorans-based transactivation assay was able to measure concentrations of about 311 ng L(-1) in the influent stream but could not detect progesterone activity in effluent. One key feature of the assay is the robustness of A. adeninivorans, which allows sample measurement without any sample preparation.

Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol.

The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200 U l(-1) ADH and 370 U l(-1) G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l(-1) ADH and 170 U l(-1) GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH-LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP(+), makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74 g l(-1) day(-1) or 406 g kg(-1) dry cell weight (dcw) day(-1) isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol product per mole NADP(+). In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.

Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans.

The yeast Arxula adeninivorans has been previously shown to secrete a large amount of an electro-active molecule. The molecule was produced by cells that had been cultivated in a rich medium, harvested, washed and then suspended in phosphate-buffered saline (PBS). The molecule was easily detectable after 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, and it was shown to be a solution species rather than a cell-attached species. We have optimised the production of the molecule, identified it by high-pressure liquid chromatography (HPLC) fractionation and high-resolution mass spectrometric analysis and determined the pathway involved in its synthesis. It has a mass/charge ratio that corresponds to uric acid, and this identification was supported by comparing UV spectra and cyclic voltammograms of the samples to those of uric acid. An A. adeninivorans xanthine oxidase gene disruption mutant failed to produce uric acid, which added further validity to this identification. It also demonstrated that the purine catabolism pathway is involved in its production. A transgenic A. adeninivorans strain with a switchable urate oxidase gene (AUOX) accumulated uric acid when the gene was switched off but did not when the gene was switched on. Cultivation of cells on amino acid and purine-free minimal media with an inorganic nitrogen source suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway.

Bioelectrochemical probing of intracellular redox processes in living yeast cells--application of redox polymer wiring in a microfluidic environment.

Conventionally, microbial bioelectrochemical assays have been conducted using immobilized cells on an electrode that is placed in an electrochemical batch cell. In this paper, we describe a developed microfluidic platform with integrated microelectrode arrays for automated bioelectrochemical assays utilizing a new double mediator system to map redox metabolism and screen for genetic modifications in Saccharomyces cerevisiae cells. The function of this new double mediator system based on menadione and osmium redox polymer (PVI-Os) is demonstrated. "Wiring" of S. cerevisiae cells using PVI-Os shows a significant improvement of bioelectrochemical monitoring in a microfluidic environment and functions as an effective immobilization matrix for cells that are not strongly adherent. The function of the developed microfluidic platform is demonstrated using two strains of S. cerevisiae, ENY.WA and its deletion mutant EBY44, which lacks the enzyme phosphoglucose isomerase. The cellular responses to introduced glucose and fructose were recorded for the two S. cerevisiae strains, and the obtained results are compared with previously published work when using an electrochemical batch cell, indicating that microfluidic bioelectrochemical assays employing the menadione-PVI-Os double mediator system provides an effective means to conduct automated microbial assays.

Assessment of Schwanniomyces occidentalis as a host for protein production using the wide-range Xplor2 expression platform.

The wide-range transformation/expression platform, Xplor2, was employed for the assessment of Schwanniomyces occidentalis as a potential producer of the recombinant proteins human IFNα2a (IFNα2a) and S. occidentalis fructofuranosidase (SFfase), and its efficiency was compared to that of Arxula adeninivorans. ADE2 and URA3 genes from both yeast species were isolated, characterized and used as selection markers in combination with the IFNα2a and SFfase expression modules, which used the strong constitutive A. adeninivorans-derived TEF1 promoter. Yeast rDNA integrative expression cassettes and yeast integrative expression cassettes equipped with a selection marker and expression modules were transformed into auxotrophic S. occidentalis and A. adeninivorans strains and a quantitative comparison of the expression efficiency was made. Whilst IFNα2a was mainly accumulated extracellularly (>95 %) in A. adeninivorans, extracellular SFfase (>90 %) was detected in both yeast species. The DNA composition of the selection marker modules and expression modules, especially their open reading frame codon usage, affects auxotrophy recovery as well as protein expression. Auxotrophy recovery was only achieved with selection marker modules of the homologous gene donor yeast. The concentration of recombinant IFNα2a was fivefold higher in A. adeninivorans (1 mg L(-1)), whereas S. occidentalis accumulated 1.5- to 2-fold more SFfase (0.5 Units ml(-1)). These results demonstrate the extension of the use of the wide-range expression platform Xplor2 to another yeast species of biotechnological interest.

Patterned forests of vertically-aligned multiwalled carbon nanotubes using metal salt catalyst solutions.

A simple method for producing patterned forests of multiwalled carbon nanotubes (MWCNTs) is described. An aqueous metal salt solution is spin-coated onto a substrate patterned with photoresist by standard methods. The photoresist is removed by acetone washing leaving the acetone-insoluble catalyst pattern on the substrate. Dense forests of vertically aligned (VA) MWCNTs are grown on the patterned catalyst layers by chemical vapour deposition. The procedures have been demonstrated by growing MWCNT forests on two substrates: silicon and conducting graphitic carbon films. The forests adhere strongly to the substrates and when grown directly on carbon film, offer a simple method of preparing MWCNT electrodes.

GiFRD encodes a protein involved in anaerobic growth in the arbuscular mycorrhizal fungus Glomus intraradices.

Fumarate reductase is a protein involved in the maintenance of redox balance during oxygen deficiency. This enzyme irreversibly catalyzes the reduction of fumarate to succinate and requires flavin cofactors as electron donors. Two examples are the soluble mitochondrial and the cytosolic fumarate reductases of Saccharomyces cerevisiae encoded by the OSM1 and FRDS1 genes, respectively. This work reports the identification and characterization of the gene encoding cytosolic fumarate reductase enzyme in the arbuscular mycorrhizal fungus, Glomus intraradices and the establishment of its physiological role. Using a yeast expression system, we demonstrate that G. intraradices GiFRD encodes a protein that has fumarate reductase activity which can functionally substitute for the S. cerevisiae fumarate reductases. Additionally, we showed that GiFRD transformants are not affected by presence of salt in medium, indicating that the presence of this gene has no effect on yeast behavior under osmotic stress. The fact that GiFRD expression and enzymatic activity was present only in asymbiotic stage confirmed existence of at least one anaerobic metabolic pathway in this phase of fungus life cycle. This suggests that the AMF behave as facultative anaerobes in the asymbiotic stage.

Role of the AFRD1-encoded fumarate reductase in hypoxia and osmotolerance in Arxula adeninivorans.

Fumarate reductase is an enzyme involved in maintaining redox balance through regeneration of reduced cofactors during oxygen deficiency conditions. This work reports the identification and characterization of the gene and its promoter and terminator elements that encodes cytosolic fumarate reductase enzyme in the nonconventional yeast, Arxula adeninivorans. The gene harbours an ORF of 1446 bp, encoding a 482-amino acid protein. The deduced amino acid sequence is similar to those of fumarate reductases from other yeast and fungi, such as the two fumarate reductases of Saccharomyces cerevisiae, Frd1p (44%) and Osm1p (41%). This enzyme is located in the cytosol and has a pH optimum of ca. 7.5 and a Michaelis constant (K(M)) of 2.9 mM with fumarate as the substrate. Expression of AFRD1 is regulated by the cultivation conditions. A shift from NaCl-free to NaCl-supplemented media and aerobic to hypoxic growth conditions leads to reduced AFRD1 transcription levels, but not to alteration in the concentration of Afrd1p. The functional analyses of Afrd1p were performed in A. adeninivorans and S. cerevisiae disruption mutants. The A. adeninivorans fumarate reductase is capable of functional complementation of the missing S. cerevisiae genes during anoxia; however, it is not involved in yeast growth under osmotic stress.

Reproducible fabrication of robust, renewable vertically aligned multiwalled carbon nanotube/epoxy composite electrodes.

We describe the reproducible fabrication of robust, vertically aligned multiwalled carbon nanotube (VACNT)/epoxy composite electrodes. The electrodes are characterized by cyclic voltammetry, impedance spectroscopy, and scanning electron and atomic force microscopies. Low background currents are obtained at the electrodes, and common redox probe molecules and NADH show excellent voltammetric behavior. When electrode performance deteriorates due to fouling, the electrode surfaces can be reproducibly renewed by mechanical polishing followed by O(2) plasma treatment. The electrochemical performance of the electrodes is maintained after more than 100 cycles of use and renewal.

Robust forests of vertically aligned carbon nanotubes chemically assembled on carbon substrates.

Forests of vertically aligned carbon nanotubes (VACNTs) have been chemically assembled on carbon surfaces. The structures show excellent stability over a wide potential range and are resistant to degradation from sonication in acid, base, and organic solvent. Acid-treated single-walled carbon nanotubes (SWCNTs) were assembled on amine-terminated tether layers covalently attached to pyrolyzed photoresist films. Tether layers were electrografted to the carbon substrate by reduction of the p-aminobenzenediazonium cation and oxidation of ethylenediamine. The amine-modified surfaces were incubated with cut SWCNTs in the presence of N,N'-dicyclohexylcarbodiimide (DCC), giving forests of vertically aligned carbon nanotubes (VACNTs). The SWCNT assemblies were characterized by scanning electron microscopy, atomic force microscopy, and electrochemistry. Under conditions where the tether layers slow electron transfer between solution-based redox probes and the underlying electrode, the assembly of VACNTs on the tether layer dramatically increases the electron-transfer rate at the surface. The grafting procedure, and hence the preparation of VACNTs, is applicable to a wide range of materials including metals and semiconductors.

Patterned arrays of vertically aligned carbon nanotube microelectrodes on carbon films prepared by thermal chemical vapor deposition.

A straightforward procedure is described for preparation of arrays of microdisk electrodes comprising bundles of vertically aligned carbon nanotubes (VACNTs). The arrays are fabricated by thermal chemical vapor deposition synthesis directly on a planar carbon film support. Use of standard micro- and nanolithography procedures for patterning the bilayer catalyst spots enables arrays to be grown with controlled electrode diameters and spacings. The minimum accessible VACNT bundle diameter, and hence microelectrode diameter, is 2 microm. After insulating the arrays with SU-8 epoxy and exposing the VACNT ends by polishing or treating with O2 plasma, the microdisk electrodes exhibit attractive electrochemical properties.

Electrochemical detection of wild type Saccharomyces cerevisiae responses to estrogens.

The presence of an estrogen binding protein (EBP) and an endogenous ligand in three yeast species was first reported in 1982/1983. The ligand was shown to be 17beta-estradiol and the binding affinities of EBP were demonstrated to be similar to those of rat estrogen receptors. This report describes detection of the behaviour of a putative estrogen binding protein in Saccharomyces cerevisiae using a double mediator electrochemical detection system. The response to estrogen is shown to be quantitative with signals detectable from 10(-8) to 10(-14)M. An incubation period of 5h is established and a method to block electrochemical signals produced by the catabolism of exogenous substrates is demonstrated to be effective. The system provides a method that permits the use of wild type S. cerevisiae to quantify estrogens.

A novel estrogen sensor based on recombinant Arxula adeninivorans cells.

A novel yeast cell-based assay was developed for the detection of estrogenic activity in wastewater. Recombinant Arxula adeninivorans strains were engineered to co-express the human estrogen receptor alpha (hERalpha) and a Klebsiella-derived phytase (phyK) reporter gene under the control of an A. adeninivorans-derived glucoamylase (GAA) promoter which had been modified by the insertion of estrogen-responsive elements (EREs). In the presence of estrogenic compounds, hERalpha dimerizes and binds to the estrogen. Reporter gene expression is induced by subsequent binding of the hERalpha-dimer/estrogen complex to estrogen responsive elements (ERE) in the promoter. The insertion of different numbers of EREs in three alternative promoter positions and its effect on reporter gene expression were assessed. In one of the constructs, a detection limit of 5 ng l(-1) and a determination limit of 10 ng l(-1) for 17beta-estradiol-like activity was achieved. The photometric assay used enabled estrogen determination in sewage samples within 30 h.

A novel enzymatic approach in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine degradative enzymes.

The purine degradation pathway in humans ends with uric acid, which has low water solubility. When the production of uric acid is increased either by elevated purine intake or by impaired kidney function, uric acid will accumulate in the blood (hyperuricemia). This increases the risk of gout, a disease described in humans for at least 1000 years. Many lower organisms, such as the yeast Arxula adeninivorans, possess the enzyme, urate oxidase that converts uric acid to 5-hydroxyisourate, thus preventing uric acid accumulation. We have examined the complete purine degradation pathway in A. adeninivorans and analyzed enzymes involved. Recombinant adenine deaminase, guanine deaminase, urate oxidase and endogenous xanthine oxidoreductase have been investigated as potential additives to degrade purines in the food. Here, we review the current model of the purine degradation pathway of A. adeninivorans and present an overview of proposed enzyme system with perspectives for its further development.

Use of recombinant oestrogen binding protein for the electrochemical detection of oestrogen.

We have previously reported the development of an electrochemical method to quantitatively detect vertebrate oestrogens using an oestrogen binding protein (EBP1) present in wild type Saccharomyces cerevisiae and Candida albicans cells. However, the assays were complex and slow with both whole cells and cell lysate. In this work we report the transfer of the EBP1 gene to an industrial yeast, the addition of a his tag sequence to simplify purification of the protein, and the oestrogen binding characteristics of the protein. The recombinant protein (Ebp1p-6h) can now be produced by a non-pathogenic cell, and has shown good stability both when in use and when lyophilised. The detection range covers likely environmental concentrations of free oestrogens and the limit of detection is below the environmental concentration that has significant biological effect. In addition the assay period has been reduced to approximately 2min. This work reports progress toward the construction of a rapid, portable oestrogen sensor that is not restricted to use to the laboratory.

MICREDOX--development of a ferricyanide-mediated rapid biochemical oxygen demand method using an immobilised Proteus vulgaris biocomponent.

Biochemical oxygen demand (BOD) is an international regulatory environmental index for monitoring organic pollutants in wastewater and the current legislated standard test for BOD monitoring requires 5 days to complete (BOD5 test). We are developing a rapid microbial technique, MICREDOX, for measuring BOD by eliminating oxygen and, instead, quantifying an equivalent biochemical co-substrate demand, the co-substrate being a redox mediator. Elevated concentrations of Proteus vulgaris, either as free cells or immobilised in Lentikat disks, were incubated with an excess of redox mediator (potassium hexacyanoferrate(III)) and organic substrate for 1h at 37 degrees C without oxygen. The addition of substrate increased the catabolic activity of the microorganisms and the accumulation of reduced mediator, which was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. The recorded currents were converted to their BOD5 equivalent with the only assumption being a fixed conversion of substrate and known stoichiometry. Measurements are reported both for the BOD5 calibration standard solution (150 mg l(-1) glucose, 150 mg l(-1) glutamic acid) and for filtered effluent sampled from a wastewater treatment plant. The inclusion of a highly soluble mediator in place of oxygen facilitated a high ferricyanide concentration in the incubation, which in turn permitted increased concentrations of microorganisms to be used. This substantially reduced the incubation time, from 5 days to 1h, for the biological oxidation of substrates equivalent to those observed using the standard BOD5 test. Stoichiometric conversion efficiencies for the oxidation of the standard substrate by P. vulgaris were typically 60% for free cells and 35-50% for immobilised cells.