Isolation and characterization of mutated alcohol oxidases from the yeast Hansenula polymorpha with decreased affinity toward substrates and their use as selective elements of an amperometric biosensor

<p>Abstract</p> <p>Background</p> <p>Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution.</p> <p>Results</p> <p><it>Hansenula polymorpha </it>(<it>Pichia angusta</it>) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the K_Mvalues for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas V_maxvalues are increased or remain unaffected. The mutant AOX alleles from <it>H. polymorpha </it>mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX.</p> <p>Conclusion</p> <p>The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids.</p

Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

<p>Abstract</p> <p>Background</p> <p>The thermotolerant methylotrophic yeast <it>Hansenula polymorpha </it>is capable of alcoholic fermentation of xylose at elevated temperatures (45 – 48°C). Such property of this yeast defines it as a good candidate for the development of an efficient process for simultaneous saccharification and fermentation. However, to be economically viable, the main characteristics of xylose fermentation of <it>H. polymorpha </it>have to be improved.</p> <p>Results</p> <p>Site-specific mutagenesis of <it>H. polymorpha XYL1 </it>gene encoding xylose reductase was carried out to decrease affinity of this enzyme toward NADPH. The modified version of <it>XYL1 </it>gene under control of the strong constitutive <it>HpGAP </it>promoter was overexpressed on a <it>Δxyl1 </it>background. This resulted in significant increase in the K_Mfor NADPH in the mutated xylose reductase (K341 → R N343 → D), while K_Mfor NADH remained nearly unchanged. The recombinant <it>H. polymorpha </it>strain overexpressing the mutated enzyme together with native xylitol dehydrogenase and xylulokinase on <it>Δxyl1 </it>background was constructed. Xylose consumption, ethanol and xylitol production by the constructed strain were determined for high-temperature xylose fermentation at 48°C. A significant increase in ethanol productivity (up to 7.3 times) was shown in this recombinant strain as compared with the wild type strain. Moreover, the xylitol production by the recombinant strain was reduced considerably to 0.9 mg × (L × h)^-1as compared to 4.2 mg × (L × h)^-1for the wild type strain.</p> <p>Conclusion</p> <p>Recombinant strains of <it>H. polymorpha </it>engineered for improved xylose utilization are described in the present work. These strains show a significant increase in ethanol productivity with simultaneous reduction in the production of xylitol during high-temperature xylose fermentation.</p

Transcriptional activator Cat8 is involved in regulation of xylose alcoholic fermentation in the thermotolerant yeast Ogataea (Hansenula) polymorpha

Additional file 5. List of primers used in this study (restriction sites are underlined)

Development of the plate assay screening procedure for isolation of the mutants deficient in inactivation of peroxisomal enzymes in the yeast Yarrowia lipolytica

The amine oxidase (AMO) plate assay screening procedure for isolation of the mutants deficient in inactivation of peroxisomal enzymes in the yeast Y. lipolytica has been developed. The first tagged mutants affected in the peroxisomal AMO and isocitrate lyase inactivation were generated by the insertion of a zeta-URA3 mutagenesis cassette into the genome of a zeta-free and ura3 deletion mutant strain of Y. lipolytica.Розроблено метод масового відбору мутантів дріжджів Y. lipolytica з пошкодженою інактивацією пероксисомних ферментів. Отримано перші мутанти з блоком інактивації пероксисомних амінооксидази та ізоцитратліази шляхом інсерції касети zeta-URA3 в геном вільного від zeta-елементів делеційного мутанта uraЗ Y. lipolytica.Разработан метод массового отбора мутантов дрожжей Y. lipolytica с поврежденной инактивацией пероксисомных ферментов. Получены первые мутанты с блоком инактивации пероксисомных аминооксидазы и изоцитратлиазы путем инсерции кассеты zeta-URA3 в геном свободного от zeta-елементов делеционного мутанта ura3 Y. lipolytica

Optimization of glutathione production in batch and fed-batch cultures by the wild-type and recombinant strains of the methylotrophic yeast Hansenula polymorpha DL-1

<p>Abstract</p> <p>Background</p> <p>Tripeptide glutathione (gamma-glutamyl-L-cysteinyl-glycine) is the most abundant non-protein thiol that protects cells from metabolic and oxidative stresses and is widely used as medicine, food additives and in cosmetic industry. The methylotrophic yeast <it>Hansenula polymorpha </it>is regarded as a rich source of glutathione due to the role of this thiol in detoxifications of key intermediates of methanol metabolism. Cellular and extracellular glutathione production of <it>H. polymorpha </it>DL-1 in the wild type and recombinant strains which overexpress genes of glutathione biosynthesis (<it>GSH2</it>) and its precursor cysteine (<it>MET4</it>) was studied.</p> <p>Results</p> <p>Glutathione producing capacity of <it>H. polymorpha </it>DL-1 depending on parameters of cultivation (dissolved oxygen tension, pH, stirrer speed), carbon substrate (glucose, methanol) and type of overexpressed genes of glutathione and its precursor biosynthesis during batch and fed-batch fermentations were studied. Under optimized conditions of glucose fed-batch cultivation, the glutathione productivity of the engineered strains was increased from ~900 up to ~ 2300 mg of Total Intracellular Glutathione (TIG) or GSH+GSSG_in, per liter of culture medium. Meantime, methanol fed-batch cultivation of one of the recombinant strains allowed achieving the extracellular glutathione productivity up to 250 mg of Total Extracellular Glutathione (TEG) or GSH+GSSG_ex, per liter of the culture medium.</p> <p>Conclusions</p> <p><it>H. polymorpha </it>is an competitive glutathione producer as compared to other known yeast and bacteria strains (<it>Saccharomyces cerevisiae, Candida utilis, Escherichia coli, Lactococcus lactis </it>etc.) with good perspectives for further improvement especially for production of extracellular form of glutathione.</p

Metabolic engineering and classical selection of the methylotrophic thermotolerant yeast Hansenula polymorpha for improvement of high-temperature xylose alcoholic fermentation

BACKGROUND: The methylotrophic yeast, Hansenula polymorpha is an industrially important microorganism, and belongs to the best studied yeast species with well-developed tools for molecular research. The complete genome sequence of the strain NCYC495 of H. polymorpha is publicly available. Some of the well-studied strains of H. polymorpha are known to ferment glucose, cellobiose and xylose to ethanol at elevated temperature (45 – 50°C) with ethanol yield from xylose significantly lower than that from glucose and cellobiose. Increased yield of ethanol from xylose was demonstrated following directed metabolic changes but, still the final ethanol concentration achieved is well below what is considered feasible for economic recovery by distillation. RESULTS: In this work, we describe the construction of strains of H. polymorpha with increased ethanol production from xylose using an ethanol-non-utilizing strain (2EthOH(−)) as the host. The transformants derived from 2EthOH(−) overexpressing modified xylose reductase (XYL1m) and native xylitol dehydrogenase (XYL2) were isolated. These transformants produced 1.5-fold more ethanol from xylose than the original host strain. The additional overexpression of XYL3 gene coding for xylulokinase, resulted in further 2.3-fold improvement in ethanol production with no measurable xylitol formed during xylose fermentation. The best ethanol producing strain obtained by metabolic engineering approaches was subjected to selection for resistance to the known inhibitor of glycolysis, the anticancer drug 3-bromopyruvate. The best mutant selected had an ethanol yield of 0.3 g/g xylose and produced up to 9.8 g of ethanol/l during xylose alcoholic fermentation at 45°C without correction for ethanol evaporation. CONCLUSIONS: Our results indicate that xylose conversion to ethanol at elevated temperature can be significantly improved in H. polymorpha by combining methods of metabolic engineering and classical selection

A Hexose Transporter Homologue Controls Glucose Repression in the Methylotrophic Yeast Hansenula polymorpha

Peroxisome biogenesis and synthesis of peroxisomal enzymes in the methylotrophic yeast Hansenula polymorpha are under the strict control of glucose repression. We identified an H. polymorpha glucose catabolite repression gene (HpGCR1) that encodes a hexose transporter homologue. Deficiency in GCR1 leads to a pleiotropic phenotype that includes the constitutive presence of peroxisomes and peroxisomal enzymes in glucose-grown cells. Glucose transport and repression defects in a UV-induced gcr1-2 mutant were found to result from a missense point mutation that substitutes a serine residue (Ser85) with a phenylalanine in the second predicted transmembrane segment of the Gcr1 protein. In addition to glucose, mannose and trehalose fail to repress the peroxisomal enzyme, alcohol oxidase in gcr1-2 cells. A mutant deleted for the GCR1 gene was additionally deficient in fructose repression. Ethanol, sucrose, and maltose continue to repress peroxisomes and peroxisomal enzymes normally and therefore, appear to have GCR1-independent repression mechanisms in H. polymorpha. Among proteins of the hexose transporter family of baker’s yeast, Saccharomyces cerevisiae, the amino acid sequence of the H. polymorpha Gcr1 protein shares the highest similarity with a core region of Snf3p, a putative high affinity glucose sensor. Certain features of the phenotype exhibited by gcr1 mutants suggest a regulatory role for Gcr1p in a repression pathway, along with involvement in hexose transport

Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX group