Volitional Qualities Development of the Students of Cooperative College.

У статті досліджено розвиток вольових якостей студентів кооперативного коледжу. The article focuses on volitional qualities development of the students of cooperative college

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

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

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Effect of Gene SFU1 on Riboflavin Synthesis in Flavinogenic Yeast Candida famata

Riboflavin or vitamin B-2 is a necessary component for all living organisms since it is the precursor of flavin coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), which are involved in numerous enzymatic reactions. Flavinogenic yeastCandida famataoverproduces riboflavin under iron starvation; however, regulation of this process is poorly understood. Regulatory gene SEF1 encoding the transcription activator has been identified. Its deletion blocks yeast's ability to overproduce riboflavin under iron starvation. It is known that, in the pathogenic flavinogenic yeastC. albicans, Sfu1 (GATA-type transcription factor) represses SEF1. It is demonstrated in this study that deletion of the SEF1 gene in wild typeC. famataleads to overproduction of riboflavin

Co-Overexpression of RIB1 and RIB6 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin or vitamin B2 is a water-soluble vitamin and a precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which play a key role as enzyme cofactors in energy metabolism. Candida famata yeast is a promising producer of riboflavin, as it belongs to the group of so-called flavinogenic yeasts, capable of riboflavin oversynthesis under conditions of iron starvation. The role of the particular structural genes in the limitation of riboflavin oversynthesis is not known. To study the impact of overexpression of the structural genes of riboflavin synthesis on riboflavin production, a set of plasmids containing genes RIB1, RIB6, and RIB7 in different combinations was constructed. The transformants of the wild-type strain of C. famata, as well as riboflavin overproducer, were obtained, and the synthesis of riboflavin was studied. It was found that overexpression of RIB1 and RIB6 genes coding for enzymes GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase, which catalase the initial steps of riboflavin synthesis, elevated riboflavin production by 13-28% relative to the parental riboflavin-overproducing strains

SEF1 and VMA1 Genes Regulate Riboflavin Biosynthesis in the Flavinogenic Yeast Candida Famata

Riboflavin (vitamin B-2) is an important component of the diet of living organisms since it is a precursor of flavin coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide) involved in numerous enzymatic reactions. It is known that flavinogenic yeastC. famatais able to perform riboflavin overproduction under conditions of iron deficiency, but the regulation of this process remains unknown. It was shown that the deletion of the SEF1 gene (encoding transcription activator) blocked the ability for riboflavin overproduction under conditions of iron deficiency. It was determined that SEF1 promoters of other flavinogenic yeasts (Candida albicansandCandida tropicalis) fused with SEF1 ORF ofC. famatacan restore the overproduction of riboflavin in the sef1 Delta mutant. The disruption of the VMA1 gene (encoding the vacuolar ATPase subunit A) led to overproduction of riboflavin in C. famatain iron complete medium