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Saccharomyces cerevisiae Aat2p contains a peroxisomal targeting signal type-1 and localizes to peroxisomes in oleate-grown cells, but not in glucose-grown cells. Here, we have investigated Aat2p from the yeast Hansenula polymorpha, which lacks a recognizable peroxisomal targeting signal. Aat2p tagged with GFP at its C terminus displays a dual cytosol-peroxisome localization in ethanol-grown cells. The partial peroxisomal localization of Aat2p persisted in the absence of the classical cycling receptors Pex5p and Pex7p but Aat2p targeting to peroxisomes was reduced in cells deleted for the matrix protein import factors PEX1, PEX2 and PEX13. Furthermore, we demonstrate that Aat2p targeting to peroxisomes requires Pex20p. Together, our data identify a Pex20p-dependent pathway for targeting Aat2p to peroxisomes.

Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature (Tm ) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.

In emergency medicine, blood lactate levels are commonly measured to assess the severity and response to treatment of hypoperfusion-related diseases (e.g., sepsis, trauma, cardiac arrest). Clinical blood lactate testing is conducted with laboratory analyzers, leading to a delay of 3 h between triage and lactate result. Here, a fluorescence-based blood lactate assay, which can be utilized for bedside testing, based on measuring the hydrogen peroxide generated by the enzymatic oxidation of lactate is described. To establish a hydrogen peroxide assay, near-infrared cyanine derivatives are screened and sulfo-cyanine 7 is identified as a new horseradish peroxidase (HRP) substrate, which loses its fluorescence in presence of HRP and hydrogen peroxide. As hydrogen peroxide is rapidly cleared by erythrocytic catalase and glutathione peroxidase, sulfo-cyanine 7, HRP, and lactate oxidase are encapsulated in a liposomal reaction compartment. In lactate-spiked bovine whole blood, the newly developed lactate assay exhibits a linear response in a clinically relevant range after 10 min. Substituting lactate oxidase with glucose and alcohol oxidase allows for blood glucose, ethanol, and methanol biosensing, respectively. This easy-to-use, rapid, and versatile assay may be useful for the quantification of a variety of enzymatically oxidizable metabolites, drugs, and toxic substances in blood and potentially other biological fluids.

Structure-guided directed evolution of choline oxidase has been carried out by using the oxidation of hexan-1-ol to hexanal as the target reaction. A six-amino-acid variant was identified with a 20-fold increased kcat compared to that of the wild-type enzyme. This variant enabled the oxidation of 10 mm hexanol to hexanal in less than 24 h with 100 % conversion. Furthermore, this variant showed a marked increase in thermostability with a corresponding increase in Tm of 20 °C. Improved solvent tolerance was demonstrated with organic solvents including ethyl acetate, heptane and cyclohexane, thereby enabling improved conversions to the aldehyde by up to 30 % above conversion for the solvent-free system. Despite the evolution of choline oxidase towards hexan-1-ol, this new variant also showed increased specific activities (by up to 100-fold) for around 50 primary aliphatic, unsaturated, branched, cyclic, benzylic and halogenated alcohols.

The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

BACKGROUND The effects of designed methanol and co-substrate feeding strategies on fed-batch recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut+ were investigated. RESULTS The highest cell concentration was obtained as 157 g L−1 with the exponential feeding strategy where methanol was fed at a pre-calculated feeding rate based on pre-determined µ0 = 0.03 h−1, and mannitol concentration was kept constant at 50 g L−1 for t=0–6 h. The highest rhGH concentration was obtained as 1.3 g L−1 at t=54 h with the methanol-stat feeding strategy, where no co-substrate was used and the methanol concentration was kept at 5 g L−1 in the medium during the production phase. Thereafter, in another methanol-stat feeding strategy with pulse-fed sorbitol, the highest rhGH concentration was obtained as 1.2 g L−1 at t=42 h. CONCLUSION The methanol-stat fed-batch method keeping methanol concentration constant at 5 g L−1 in the production medium increased rhGH production at least 2-fold compared with that of the exponential feeding strategies. In the designed methanol-stat feeding strategies, use of sorbitol as co-substrate decreased methanol demand of the cells 2-fold, increased the overall product yield on the substrate 1.6-fold, shortened the cultivation time by 12 h and increased the productivity. Thus, methanol-stat feeding with co-substrate sorbitol was found to be a favorable fed-batch strategy for rhGH production by Pichia pastoris under alcohol oxidase I promoter. © 2014 Society of Chemical Industry

The high production cost of cellulase is one of the limitations that hinder the commercialization of lignocellulose-based biorefineries. As one of the important cellulases, Neurospora crassa cellulase is not so intensively investigated as T. reesei cellulase. In this study, the cbh1gene (NCU07340) cloned from N. crassa was expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promotor. Six transformants with the highest resistance to G418 were selected by two rounds of transformant screening, among which the most robust producer of the recombinant cellobiohydrolase I (CBHI) has an Avicelase activity of 0.22 U/mL. After fermentation optimization, it was improved to 0.30 U/mL. Interestingly, the optimal temperature and pH of the recombinant CBHI were 60°C and 7.2, respectively, and it was quite stable within the wide ranges of temperature and pH. This work is a good example for the future improvement and optimization of N. crassa cellulase.

BACKGROUND Achievement of very high cell concentration is the prerequisite for enhanced porcine interferon-α (pIFN-α) production by Pichia pastoris, but it is closely associated with severe ethanol accumulation, leading to instability in pIFN-α expression. RESULTS By analyzing the transcriptional levels of genes encoding the key enzymes in methanol metabolism, it was found that high (more than 6 g L−1) and long term (more than 4 h) ethanol accumulation in the late glycerol feeding cultivation phase irreversibly repressed alcohol oxidase (AOX) promoter, leading to instability in pIFN-α production. A novel improved DO-Stat glycerol feeding strategy based on on-line ethanol measurement was thus proposed to control ethanol concentration at a low level of 2 g L−1, while maintaining cell growth at comparably high level. With the aid of the proposed strategy, maximum pIFN-α concentration increased 29.8–75.5% compared with the best one adopting the ‘traditional’ DO-Stat strategy, when the same methanol induction strategy (methanol/sorbitol co-feeding ratio of 1:1) was applied. CONCLUSION The proposed improved DO-Stat strategy is effective in simultaneously achieving very high concentration of the cells with functional cellular skeletons and repressing ethanol accumulation in the glycerol feeding phase, which enhanced and stabilized pIFN-α production during the induction condition in turn. © 2013 Society of Chemical Industry

Certain lipases such as Candida antarctica lipase A (CAL-A) are known to possess acyltransferase activity, which allows ester synthesis even in the presence of water. Discovery of novel lipases and identification of lipase variants with improved acyltransferase activity is laborious using standard methods such as gas chromatography. To overcome this limitation, an enzyme cascade was established based on quantification of the alcohol consumed in fatty acid ester synthesis from plant oils. For this, an alcohol oxidase converts the remaining alcohol to the corresponding aldehyde with concomitant formation of hydrogen peroxide. This is then quantified by the action of horseradish peroxidase leading to the oxidation of the co-substrate ABTS, which is determined spectrophotometrically at 695 nm. The conversions thus determined correspond well to data measured by off-line gas chromatography analysis and the assay could be adapted to the microtiterplate format. Practical applications: Fatty acid esters are valuable compounds in various industrial applications, i.e., fatty acid methyl esters are used as biofuels and ethyl esters can serve as fragrance or flavoring compounds in the cosmetic industry. The enzymatic synthesis of these compounds is possible, but often enzymes need to be adapted to meet process requirements. This assay simplifies the identification of suitable enzymes. Moreover, substrate specificities of a lipase for a certain alcohol or acyl acceptor can also be easily studied with this assay.

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol-induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one-compartment system cannot boost productivity for the model system under study. Hence, a two-compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen-related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two-fold increase in the titer and productivity and three-fold increase in protein-specific activity were achieved. Hence, partial application of oxygen-related stress in the two-compartment system was proposed as a process technology to enhance protein production.

The high-level expression of the xylanase GH11 gene from Aspergillus niger IA-001 called xynB was successfully completed in Pichia pastoris. The xynB gene encoding a mature xylanase of 225 amino acid was subcloned into the pPICZαA vector and was transformed into P. pastoris X-33 under the control of the alcohol oxidase I (AOX1) promoter. The xynB gene was ligated with a sequence encoding modified α-factor signal peptide (pPICZαmA) and the recombinant xylanase activity, which was measured 1280 U ml(-1), was 1.5-fold higher than when it was inserted into pPICZαA and was 19.39-fold greater than the native xylanase in the original strain. In a 10 L fermenter, the recombinant xylanase activity measured 10,035 U ml(-1) after 114 h. The SDS-PAGE analysis revealed that the purified xynB protein migrated as a single band with an apparent molecular weight of 24 kDa. The specific activity, using beechwood xylan as a substrate, was 1916 U mg(-1). The xylanase activity was optimal at pH 5.0 and at 50 °C. In addition, the xynB was active over a pH range of 2.2 to 10.0. The apparent Km and Vmax values were 4.429 mg ml(-1) and 1429 U mg(-1), respectively.

Amination of non-activated aliphatic fatty alcohols to the corresponding primary amines was achieved through a five-enzyme cascade reaction by coupling a long-chain alcohol oxidase from Aspergillus fumigatus (LCAO_Af) with a ω-transaminase from Chromobacterium violaceum (ω-TA_Cv). The alcohol was oxidized at the expense of molecular oxygen to yield the corresponding aldehyde, which was subsequently aminated by the PLP-dependent ω-TA to yield the final primary amine product. The overall cascade was optimized with respect to pH, O2 pressure, substrate concentration, decomposition of H2O2 (derived from alcohol oxidation), NADH regeneration, and biocatalyst ratio. The substrate scope of this concept was investigated under optimized conditions by using terminally functionalized C4-C11 fatty primary alcohols bearing halogen, alkyne, amino, hydroxy, thiol, and nitrile groups.

Methanol-inducible gene promoters in methanol-utilizing yeasts are used in high-level heterologous gene expression systems. Generally, expression of methanol-inducible genes is completely repressed by the presence of glucose. In this study we identified the MIG1 gene in Candida boidinii, which encodes a homologue of the glucose repressor Mig1p of Saccharomyces cerevisiae. Disruption of the CbMIG1 gene had no growth effect on various carbon sources. Activation of the methanol-inducible AOD1 gene, which encodes alcohol oxidase, was increased in the early stage of methanol induction when cells of the CbMIG1-disrupted strain were transferred from glucose medium to methanol medium. Furthermore, CbMig1p tagged with yellow fluorescent protein was primarily localized in the nucleus of glucose-grown cells, but was diffuse in the cytosol of methanol-grown cells. This cytosolic diffusion in methanol-grown cells occurred in a CbMsn5p-dependent manner. These results suggest that CbMig1p is involved in negative regulation of methanol-inducible gene expression in C. boidinii. Copyright © 2012 John Wiley & Sons, Ltd.

Hydrophobins are small surface-active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor-made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3-phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha-factor prepro-peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre-purification using foam separation.

Alcohol intoxication causes serious diseases, whereas current treatments are mostly supportive and unable to remove alcohol efficiently. Upon alcohol consumption, alcohol is sequentially oxidized to acetaldehyde and acetate by the endogenous alcohol dehydrogenase and aldehyde dehydrogenase, respectively. Inspired by the metabolism of alcohol, we develop a hepatocyte–mimicking antidote for alcohol intoxication through the co–delivery of the nanocapsules of alcohol oxidase (AOx), catalase (CAT), and aldehyde dehydrogenase (ALDH) to the liver, where AOx and CAT catalyze the oxidation of alcohol to acetaldehyde, while ALDH catalyzes the oxidation of acetaldehyde to acetate. Administered to alcohol–intoxicated mice, the antidote rapidly accumulates in the liver and enables a significant reduction of the blood alcohol concentration. Moreover, blood acetaldehyde concentration is maintained at an extremely low level, significantly contributing to liver protection. Such an antidote, which can eliminate alcohol and acetaldehyde simultaneously, holds great promise for the treatment of alcohol intoxication and poisoning and can provide therapeutic benefits.

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (A OX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine-modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at -0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4 degrees C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 896-906, 2010

Several methods for the quantitative detection of different compounds, e.g., L-amino acids, sugars or alcohols in liquid media were developed by application of an automatic measuring unit including a fluid chip-calorimeter FCC-21. For this purpose, enzymes were immobilized covalently on the inner and outer surface of CPG (controlled porous glass)-spherules with an outer diameter of 100 μm and filled into a micro flow-through reaction chamber (V R = 20 μL). The design of the measuring cell allows for easy insertion into the calorimeter device of a stored series of comfortably pre-fabricated measuring cells. These cells can be filled with different enzyme immobilizates. Different oxidases were used and co-immobilized with catalase for the improvement of the detection sensitivity. A signal amplification could be achieved up to a factor of 3.5 with this configuration. β-D-glucose, ethanol and L-lysine could be detected in a range of 0.25-1.75 mM using glucose oxidase, alcohol oxidase and lysine oxidase. The group of oxidases in combination with the enzymatic catalysis of the intermediate H 2 O 2 allows the quantitative detection of a large number of analytes. A good measurement and storage stability could be achieved for several weeks by this immobilization method. In addition to enzyme-based detection reactions, it was shown that living microorganisms can be immobilized in the reaction chamber. Thus, the system can be used as a whole-cell biosensor. The quantitative detection of phenol in the range of 10-100 μM could be performed using the actinomycete Rhodococcus sp. immobilized on glass beads by means of embedding into polymers.

The Lotus japonicus EST database was searched against Arabidopsis thaliana AtFAO3, a full-length cDNA that encodes a membrane-bound, flavin-containing, hydrogen peroxide generating, long-chain fatty alcohol oxidase. One EST fragment was detected, and the corresponding full-length cDNA was obtained by screening a cDNA library of L. japonicus. The LjFAO1 genomic DNA was amplified by PCR, to give a product 3.6 kb in length. Comparison between the LjFAO1 cDNA and genomic DNA revealed that the LjFAO1 contains 3 exons and 2 introns. RT-PCR analysis showed that the LjFAO1 was expressed in the whole plant, with the highest expression level in the apex and the lowest expression level in the siliques. The LjFAO1 gene was down-regulated by cold stress in both the apex and the cotelydon of the 8-day old seedlings, the first time that a long-chain alcohol oxidase has been shown to respond directly to stress. The full length cDNA and a C-terminal truncated version were overexpressed in Escherichia coli. The full length version of LjFAO1 exhibited long-chain fatty alcohol oxidase activity and was subsequently purified by Ni-NTA chromatography. The active LjFAO1 protein showed substrate specificities toward 1-dodecanol, 1-hexadecanol, and 1,16-hexadecanediol with Km values 59.6 +/- 14.8 (microM), 40.9 +/- 8.2 (microM) and 19.4 +/- 1.5 (microM), respectively, suggesting apparent differences in substrate preferences with AtFAO3.

Bleaching detergent formulations contain environmentally unfriendly bleaching agents (perborates and percarbonates), which cause aquatic eutrophication, although without these compounds detergents are much less efficient for the washing processes. In an effort to replace these compounds, in this study, hydrogen peroxide was generated as a bleaching compound by means of enzymatic reactions. Three different pathways were investigated. The first one was the H2O2 production from glucose by glucose oxidase. The second one was the production of H2O2 from carboxymethylcellulose (CMC) by the action of both cellulase, which promotes the hydrolysis of the polymeric chain, and glucose oxidase, which oxidizes the smaller fractions to produce H2O2. Finally, H2O2 was also obtained from ethanol, which is present in liquid detergents, by the action of the enzyme alcohol oxidase. In the search for maximal peroxide production, substrate concentration and enzymatic activities were optimized. The effect of H2O2 produced in the washing process was simulated by means of a process of cotton bleaching. Although enzymatic-reaction oxidations produced higher levels of hydrogen peroxide (up to 1 g/L after 8 h), higher improvement of cotton whiteness was achieved from CMC and from ethanol. The milder conditions of temperature and pH, biodegradability and less consumption of water and energy are advantageous for enzymes as good substitutes for H2O2 precursors and make them appropriate to be considered in detergent formulations. These enzymes could be combined with other oxidative enzymes, such as peroxidases, in order to lower the required temperature and use a pH close to the neutral value during the bleaching processes.

The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system.

This work extensively covers the use of layer-by-layer (LbL) assembly of polyelectrolytes for the transduction of catalytic and affinity events. It is demonstrated that by controlling the charge of polyelectrolytes, electrostatic interactions drive then the LbL construction of supramolecular architectures with improved performance. Particularly, by adequately charging an osmium based redox polymer, a multicofactor protein (fructose dehydrogenase) may be deposited in a favored orientation resulting in a more efficient electrochemical communication. A H2O2 transducing interface is created by the enhancement of electrostatic interactions between the electrochemical and catalytic layer. Further assemblies include the coupling of an oxidase (alcohol oxidase) with the H2O2 transducing interface resulting in a linear increase of current with the number of AOX layers. Finally, affinity assemblies are demonstrated by deposition of anti Dexamethasone antibodies. Faraday currents are then obtained by the electrochemical communication between the HRP labeled immunoconjugate and the electrochemical interface in a heterogeneous competitive assay format.

An ethanol chip biosensors based on bienzymatic system has been developed. Horse radish peroxidase and alcohol oxidase have been co-immobilized into a polypyrrole matrix, as well as the mediator, onto the integrated working electrode. Variables that affect to the chronoamperometric response of ethanol have been optimized through the experimental design methodology. Under these conditions, the slopes of several calibrations curves show a reproducibility, repeatability and limit of detection of 6.09% (n ¼ 5), 9.03% (n ¼ 5) and 2.98 � 0.38mmol dm � 3 (a ¼ b ¼ 0.05, n ¼ 3), respectively. Finally, the biosensors based on platinum chips were applied to the determination of ethanol in white wine samples, obtaining successful results.

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, PMOD1 and PMOD2, were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the PMOD1 and PMOD2 promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. PMOD1 was induced not only by methanol but also by glycerol. PMOD2 was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that PMOD2 is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by PMOD1 and PMOD2. PMOD1 expression was optimal at low methanol concentrations, whereas PMOD2 was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both PMOD2 and PMOD1 should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using PMOD1 and PMOD2 in the same host cell and in the same flask. Copyright © 2006 John Wiley & Sons, Ltd.

The hallmark of eukaryotic cells is compartmentalization of distinct cellular functions into specific organelles. This necessitates the cells to run energetically costly mechanisms to precisely control maintenance and function of these compartments. One of these continuously controls organelle activity and abundance, a process termed homeostasis. Yeast peroxisomes are favorable model systems for studies of organelle homeostasis because both the proliferation and degradation of these organelles can be readily manipulated. Here, we highlight recent achievements in regulation of peroxisome turnover in yeast, in particular Hansenula polymorpha, with a focus on directions of future research. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

In the last two decades, much progress has been made in understanding the process of induction and biogenesis of peroxisomes, essential organelles in all eukaryotes. Only relatively recently, the first molecular studies on the selective degradation of this important organelle-a process known as pexophagy, which occurs when the organelles have become redundant-have been performed, especially using methylotrophic yeasts. The finding that pexophagy and other transport pathways to the vacuole (vacuolar protein sorting, autophagy, cytoplasm-to-vacuole-targeting and endocytosis) utilize common but also unique genes has placed pexophagy in the heart of the machinery that recycles cellular material. The quest is now on to understand how peroxisome degradation has become such a highly selective process and what the signals are that trigger it. In addition, because the prime determinant of pexophagy is located on the peroxisome itself, it has become essential to study the role of peroxisomal membrane proteins in the degradation process in detail. This review highlights the main achievements of the last years. (C) 2003 Wiley-Liss, Inc.

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.

In yeast, peroxisomes are the site of specific catabolic pathways that characteristically include hydrogen peroxide producing oxidases and catalase. During the last 10 years, much progress has been made in unravelling the molecular mechanisms involved in the biogenesis of this organelle. At present, 23 different genes (PEX genes) have been identified that are involved in different aspects of peroxisome biogenesis (e.g., proliferation, formation of the peroxisomal membrane, import of matrix proteins). The principles of peroxisome degradation are still much less understood. Recently, the first yeast mutants affected in this process have become available and used to clone corresponding genes by functional complementation. In this paper, an overview is presented of the research on yeast peroxisomes, focusing on recent achievements in the molecular aspects of peroxisome development, function, and turnover. (C) 2000 Wiley-Liss. Inc.

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.

Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde‐3‐phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N‐glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The kcat and Km values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site‐directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.

Cell-free extract prepared from methanol-grown cells of the methylotrophic yeast Pichia methanolica showed nine multiple alcohol oxidase (AOD) bands on active staining in native polyacrylamide gel electrophoresis. Their molecular basis was investigated and two AOD-encoding genes, MOD1 and MOD2, were cloned from P. methanolica genome. When the two genes were expressed in a heterologous host, an alcohol oxidase-depleted strain of Candida boidinii(aod1Delta strain), both MOD1 and MOD2 partially complemented growth defect of the host strain on methanol. While expression of either MOD1 or MOD2 in C. boidinii aod1Delta strain gave a single AOD band corresponding to the band with the largest and smallest mobility among the nine AOD bands, respectively, co-expression of MOD1 and MOD2 resulted in multiple band formation. Mixed oligomerization of Mod1p and Mod2p in vitro also gave nine multiple bands. From these results, we concluded that the nine multiple forms of AOD observed on native-PAGE represent two homo-octamers and seven hetero-octamers of Mod1p and Mod2p. Using this zymogram analysis, we also found that Mod1p was preferably produced at low methanol concentrations in the media, while Mod2p was produced at higher methanol concentrations. This shows distinct regulatory features of the two AOD-encoding genes in this methylotrophic yeast.

After induction of alcohol oxidase, active Pichia pastoris cells were grown in model alcohol solutions and orange aroma, a by-product rich in potential alcohol substrates for bio-oxidation to aldehydes. Acetaldehyde production efficiency was 60% at 0.1% ethanol, decreasing with increasing ethanol. Acetaldehyde, propanal, and octanal were generated using the Pichia system with orange aroma as substrate. Acetaldehyde concentration doubled during a 24h reaction time at 30°C. In contrast to use of isolated alcohol oxidase, the whole cell system had the advantage of cofactor regeneration and associated enzyme systems, including formaldehyde dehydrogenase. Sensory differences were found in the full strength biomodified aroma. Presented in a paired comparison aroma test, to a trained panel it rated higher for the attributes orange character, freshness, and fruitiness than the unmodified control.

The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions: FAD-binding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain. The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain. In addition, the roles of conserved residues are discussed wherever possible.

We describe a protein expression system in the methylotrophic yeast, Pichia methanolica. Methods for transformation and genetic manipulation of the organism were developed using an ade2 strain and the wild-type ADE2 gene. A vacuolar protease-deficient strain was constructed. Two genes encoding alcohol oxidases were found, yet a single isoform of alcohol oxidase was produced during methanol-fed fermentations. The promoter from this gene was used to drive expression. An integrating plasmid for the cytoplasmic expression of the 65 kDa isoform of human glutamate decarboxylase (human GAD65) was assembled. A strain harboring eight copies of this plasmid expressed enzymatically active human GAD65 at levels approaching 0.5 g/l. Identical amounts were made in Pichia pastoris. The recombinant GAD65 was purified to greater than 90% purity.

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol.

An enzyme biosensor specific for formaldehyde has been developed using pH-sensitive field effect transistors (FET) as a transducer and alcohol oxidase (AOX) from Hansenula polymorpha as a sensitive element. The steady-state response time of the developed biosensor was 10–60 s. The linear dynamic range (in semi-logarithmic scale) of the sensor output signal corresponded to 10–300 mmol dm−3 formaldehyde concentration. The operational stability of the biosensor was not less than 7 h and the relative standard deviation approximately 1–3%. When stored at +4°C, the response was stable for more than 30 days. The biosensor demonstrated high specificity to formaldehyde with no potentiometric response to primary alcohols. The influence of buffer concentration and enzyme concentration in the biomembrane on the sensor signal was investigated. The specific biosensor could also be successfully used for highly selective, quantitative estimation of mercury ion in an aqueous solution. © 1997 SCI.

Bioelectrochemical 2-Propanol Biosensors 2-Propanol biosensors for O2-detectors based on bienzyme membranes with sequentially installed catalase and alcohol oxidase are described. Measurements were performed in undiluted media up to 20 vol.-% 2-propanol according to the principle of intermediate carrier analysis by use of a micro-dialysator, an oxygenating pump and a pressure adjusting chamber. For the extension of the measuring scale in the enzyme membrane O2 is released out of hydrogen peroxide within the phosphate buffered carrierstream by catalase. Therefore, oxygen is available to the serially installed alcohol oxidase for the enzymatic substrate transformation beyond the physically soluted part of oxygen in the measuring medium. Molecular selectivities of bienzyme membranes are described for normally used moisturizing concentrates in printeries.

Protein multilayers composed of avidin and biotin-labeled alcohol oxidase (AOx) were prepared on the surface of a platinum (Pt) electrode by repeated deposition of the proteins to develop amperometric alcohol sensors. The AOx multilayer-modified sensors showed a rapid response to ethanol over the concentration range of 0.01–10mg/100mL. The output current of the sensor depended linearly upon the thickness of the AOx layers (or the number of the AOx layers), suggesting that a constant amount of AOx can be immobilized by each deposition. The selectivity of the sensor to aliphatic alcohols was in the order of methanol > ethanol > n-propanol > n-butanol, being in accord with the selectivity of AOx in a homogeneous system itself. The sensor retained its original response to ethanol for about one month without deterioration.

A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.

A series of hollow fiber membranes containing immobilized enzymes were prepared and used in the synthesis of 11 C-labelled nucleosides. 11 C-Formaldehyde was produced in an alcohol oxidase/catalase bioreactor and circulated through a thymidylate synthase bioreactor with an appropriate substrate to produce the corresponding 11 C-nucleotide. These labelled nucleotides were subsequently dephosphorylated in an alkaline phosphatase bioreactor. The bioreactor approach was amenable to hot-cell conditions and yielded 11 C-products in higher yield and shorter synthesis times than conventional chemical approaches.

Hydrogen peroxide generating alcohol oxidase (membrane bound) and aldehyde oxidase (cytosolic) were detected in tissues of the giant African land snail Achatina fulica. The highest specific activities were found in the kidney followed by the digestive gland. Solubilised digestive gland alcohol oxidase had a pH optimum of approximately 7.2. The enzyme oxidises a broad range of aromatic and aliphatic (C2C16) primary alcohols. Subcellular fractionation of digestive gland homogenate indicates that alcohol oxidase is most likely located in the endoplasmic reticulum. Cytosolic aldehyde oxidase was active with aromatic and aliphatic aldehyde but is not active with purines. A digestive role is suggested for these two enzymes. © Wiley-Liss, Inc.

Alcohol oxidase-catalyzed oxidation of benzyl alcohol to benzaldehyde was studied in three two-liquid phase systems involving butyl acetate, xylene and n-decane. A more highly hydrophobic solvent resulted in more favorable partitioning of the substrate into the aqueous phase and less deleterious effect on the enzyme activity. As a result, the overall production of aldehyde was highest with the n-decane system and lowest with the butyl acetate system. In addition, the organic solvent served as an extractant for the product, which acted both as a competitive inhibitor and as a denaturant of the enzyme. The time courses of benzaldehyde production were simulated by a simple mathematical model assuming Michaelis–Menten kinetics in the aqueous phase, enzyme deactivation by organic solvents and the equilibrium partitioning of the reagents between the two phases.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43-degrees-C but grow well at 35-degrees-C. In both strains peroxisomes were completely absent in cells grown at 43-degrees-C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive' growth conditions (less-than-or-equal-to 37-degrees-C). However, at intermediate temperatures (38-42-degrees-C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one-or infrequently a few-small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.

Single-layer and bilayer bienzyme electrodes based on the combination of a three-dimensional (3-D) redox epoxy network that electrically connects redox centers of bound horseradish peroxidase (HRP) to electrodes with a hydrogen peroxide generating enzyme, the redox centers of which are not connected to the redox-epoxy network, are described. In the single-layer electrodes, H2O2 generated by the first enzyme oxidizes the second enzyme HRP, which oxidizes the redox polymer network that is electrochemically reduced at 0 mV saturated calomel electrode (SCE). When the redox centers of the H2O2 generating enzyme are also electrically connected to the redox-epoxy network, the substrate reduced redox centers are oxidized by the redox polymer network, thus lowering the cathodic current. Such attenuation is avoided in bilayer electrodes, where the H2O2 producing enzyme and the redox-epoxy-HRP network are not electrically connected. The single-layer bienzyme electrodes extend the range of amperometric biosensors based on directly redox-epoxy “wired” enzymes to enzymes that are difficult to electrically connect to redox polymer networks and whose preferred or only cosubstrate is oxygen. For the difficult to wire enzyme-choline oxidase, the cathodic current density in the single-layer peroxidase and choline oxidase containing electrode is 80 μA cm−2 at 10 mM choline concentration, while the anodic current density of the directly wired enzyme is only 5 μA cm−2. Alcohol oxidase is not electrically connected to the wiring 3-D redox-epoxy network. The anodic current density of its redox-epoxy wired electrodes is close to nil, while the cathodic current density, observed in alcohol oxidase and wired peroxidase containing single-layer electrodes at 10 mM ethanol, is 5 μA cm−2. When well-wired enzymes, such as glucose oxidase or lactate oxidase, are utilized in single-layer electrodes, limiting cathodic current densities of 60 μA cm−2 are observed for both enzymes. These currents are much lower than those observed in the directly wired enzyme anodes.

We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous culture on a glucose/methanol mixture, indicated that various PMPs were normally synthesized. As in wild type (WT) cells, the levels of PMP synthesis appeared to be dependent on specific cultivation conditions, e.g. the carbon source used for growth. In contrast to WT controls, PMPs in methanol-induced per mutants were not subject to proteolytic degradation. Biochemical and immuno(cyto)chemical studies suggested that the PMPs in methanol-induced per cells were located in small proteinaceous aggregates, separated from peroxisomal matrix proteins that were also present in the cytosol. Vesicular membranous structures, resembling the morphology of intact peroxisomes, were never detected irrespective of the growth conditions employed.

We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg-ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSCI and hsp60, respectively, from S. cerevisiae.

Alcohol oxidase (MOX), a major peroxisomal protein of methanol-utilizing yeasts, contains two different forms of flavin adenine dinucleotide, one of which is identical with natural FAD whereas the other (mFAD) is a stereochemical modification of the natural coenzyme. This modifictaion occurs spontaneously with FAD (but not FADH) bound to alcohol oxidase. mFAD was degraded with diphosphatase to provide authentic AMP and mFMN. The latter was degraded further with phosphtase to m-riboflavin. Analysis by 1H and 13C NMR spectroscopy of mFAD revealed that the isoalloxazide and adenine rings were intact and not modified structurally. However, significant differences were observed in the proton spectra in the sugar chains attached to the isoalloxazine ring (ribitol in the case of FAD). Similar observations were made for mFMN and m-riboflavin. Most striking in COSY spectra is the virtual absence of coupling between protons 2′ and 3′ in the sugar chain attached to the isoalloxazine ring, whereas this coupling is strong in the natural materials. However, the nature of the coupling of proton 2′ to protons 1a′ and 1b′ of the sugar chain is different in modified material. All these observations are consistent with the hypothesis that in modified cofactor the absolute configuration of carbon 2′ of the sugar chain attached to the isoalloxazine ring has changed from R to S. This indicates the presence of an arabityl sugar chain rather than the ribitol present in natural FAD. A possible mechanism for this conversion is suggested.

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition or GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition or GroFS.