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

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

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)

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

Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast

Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering

Construction of uricase-overproducing strains of Hansenula polymorpha and its application as biological recognition element in microbial urate biosensor

<p>Abstract</p> <p>Background</p> <p>The detection and quantification of uric acid in human physiological fluids is of great importance in the diagnosis and therapy of patients suffering from a range of disorders associated with altered purine metabolism, most notably gout and hyperuricaemia. The fabrication of cheap and reliable urate-selective amperometric biosensors is a challenging task.</p> <p>Results</p> <p>A urate-selective microbial biosensor was developed using cells of the recombinant thermotolerant methylotrophic yeast <it>Hansenula polymorpha </it>as biorecognition element. The construction of uricase (UOX) producing yeast by over-expression of the uricase gene of <it>H. polymorpha </it>is described. Following a preliminary screening of the transformants with increased UOX activity in permeabilized yeast cells the optimal cultivation conditions for maximal UOX yield namely a 40-fold increase in UOX activity were determined.</p> <p>The UOX producing cells were coupled to horseradish peroxidase and immobilized on graphite electrodes by physical entrapment behind a dialysis membrane. A high urate selectivity with a detection limit of about 8 μM was found.</p> <p>Conclusion</p> <p>A strain of <it>H. polymorpha </it>overproducing UOX was constructed. A cheap urate selective microbial biosensor was developed.</p

Metabolic engineering of non-conventional yeasts for construction of the advanced producers of biofuels and high-value chemicals

Non-conventional yeasts, i.e. yeasts different from Saccharomyces cerevisiae, represent heterogenous group of unicellular fungi consisting of near 1500 species. Some of these species have interesting and sometimes unique properties like ability to grow on methanol, n-alkanes, ferment pentose sugars xylose and l-arabinose, grow at high temperatures (50°С and more), overproduce riboflavin (vitamin B2) and others. These unique properties are important for development of basic science; moreover, some of them possess also significant applied interest for elaboration of new biotechnologies. Current paper represents review of the recent own results and of those of other authors in the field of non-conventional yeast study for construction of the advanced producers of biofuels (ethanol, isobutanol) from lignocellulosic sugars glucose and xylose or crude glycerol (Ogataea polymorpha, Magnusiomyces magnusii) and vitamin B2 (riboflavin) from glucose and cheese whey (Candida famata)

Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione.

The ability of baker's yeast Saccharomyces cerevisiae and of the thermotolerant methylotrophic yeast Hansenula polymorpha to produce ethanol during alcoholic fermentation of glucose was compared between wild-type strains and recombinant strains possessing an elevated level of intracellular glutathione (GSH) due to overexpression of the first gene of GSH biosynthesis, gamma-glutamylcysteine synthetase, or of the central regulatory gene of sulfur metabolism, MET4. The analyzed strains of H. polymorpha with an elevated pool of intracellular GSH were found to accumulate almost twice as much ethanol as the wild-type strain during glucose fermentation, in contrast to GSH1-overexpressing S. cerevisiae strains, which also possessed an elevated pool of GSH. The ethanol tolerance of the GSH-overproducing strains was also determined. For this, the wild-type strain and transformants with an elevated GSH pool were compared for their viability upon exposure to exogenous ethanol. Unexpectedly, both S. cerevisiae and H. polymorpha transformants with a high GSH pool proved more sensitive to exogenous ethanol than the corresponding wild-type strains.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe