Establishment of a yeast platform strain for production of p-coumaric acid through metabolic engineering of aromatic amino acid biosynthesis

Aromatic amino acids are precursors of numerous plant secondary metabolites with diverse biological functions. Many of these secondary metabolites are already being used as active pharmaceutical or nutraceutical ingredients, and there are numerous exploratory studies of other compounds with promising applications. p-Coumaric acid is derived from aromatic amino acids and, besides being a valuable chemical building block, it serves as precursor for biosynthesis of many secondary metabolites, such as polyphenols, flavonoids, and some polyketides. Here we developed a p-coumaric acid-overproducing Saccharomyces cerevisiae platform strain. First, we reduced by-product formation by knocking out phenylpyruvate decarboxylase ARO10 and pyruvate decarboxylase PDC5. Second, different versions of feedback-resistant DAHP synthase and chorismate mutase were overexpressed. Finally, we identified shikimate kinase as another important flux-controlling step in the aromatic amino acid pathway by overexpressing enzymes from Escherichia coli, homologous to the pentafunctional enzyme Aro1p and to the bifunctional chorismate synthase-flavin reductase Aro2p. The highest titer of p-coumaric acid of 1.93\ub10.26gL-1 was obtained, when overexpressing tyrosine ammonia-lyase TAL from Flavobacterium johnsoniaeu, DAHP synthase ARO4K229L, chorismate mutase ARO7G141S and E. coli shikimate kinase II (aroL) in δpdc5δaro10 strain background. To our knowledge this is the highest reported titer of an aromatic compound produced by yeast. The developed S. cerevisiae strain represents an attractive platform host for production of p-coumaric-acid derived secondary metabolites, such as flavonoids, polyphenols, and polyketides

Consideration of unresolved binaries with evaluation of the mass of star clusters

При различных предположениях о функции масс компонент двойных систем оценивается, как наличие неразрешенных двойных систем влияет на определение массы скопления.An influence of the presence of unresolved binaries onto star cluster mass estimation is evaluated with different assumptions on the mass function of binary components.Часть работ проведена при финансовой поддержке государства в лице Министерства образования и науки Российской Федерации (базовая часть госзадания, РК №AAAA-A17-117030310283-7), а также при финансовой поддержке Правительства РФ (постановление № 211, контракт № 02.A03.21.0006)

Современные концепции управления высшим учебным заведением

Целью и задачами статьи является исследование современных подходов к управлению вузом, их критический анализ и возможность оптимизации процессов деятельности вуза

Label-free electrochemical monitoring of DNA ligase activity

This study presents a simple, label-free electrochemical technique for the monitoring of DNA ligase activity. DNA ligases are enzymes that catalyze joining of breaks in the backbone of DNA and are of significant scientific interest due to their essential nature in DNA metabolism and their importance to a range of molecular biological methodologies. The electrochemical behavior of DNA at mercury and some amalgam electrodes is strongly influenced by its backbone structure, allowing a perfect discrimination between DNA molecules containing or lacking free ends. This variation in electrochemical behavior has been utilized previously for a sensitive detection of DNA damage involving the sugar-phosphate backbone breakage. Here we show that the same principle can be utilized for monitoring of a reverse process, i.e., the repair of strand breaks by action of the DNA ligases. We demonstrate applications of the electrochemical technique for a distinction between ligatable and unligatable breaks in plasmid DNA using T4 DNA ligase, as well as for studies of the DNA backbone-joining activity in recombinant fragments of E. coli DNA ligase

Использование пуллуланазы в качестве биокатализатора процесса гидролиза крахмала. Часть 1. Изучение действия пуллуланазы на амилопектиновый кукурузный крахмал

The use of debranching enzymes in starch hydrolysis is a topical direction for obtaining new types of starch products with controlled properties and a potential for the further use. The aim of the work was to study an effect of pullulanase (EC3.2.1.41) on maize amylopectin starch in the native and gelatinized state. The objects of the research were maize amylopectin starch and enzyme preparation Promozyme D6 (Novozymes, Denmark). High-performance liquid chromatography (HPLC) was used to determine the carbohydrate composition of hydrolysates. The mass fraction of reducing substances (RS) was determined by the Lane and Eynon method. A rotational viscometer was used to measure dynamic viscosity of the starch hydrolysis products. It was found that analyzed starch in the native state showed low enzymatic sensitivity to the action of pullulanase with insignificant changes in viscosity, solubility and iodine binding capacity of the samples. Pullulanase showed the highest effect on gelatinized starch during the first eight hours of incubation. After eight hours, the maximum degree of starch hydrolysis by pullulanase at a dose of 10 units/g dry matter (DM) was 4.7% on DM basis, iodine binding capacity of the hydrolysate was D600 = 0.343 (in the control experiment D600 = 0.154), and the viscosity of the hydrolysate decreased from 7887 mPa · s to 4.3 mPa · s. Hydrolysates cooled to 8 °C and held for 20 hours along with hydrolysates that were not cooled showed high susceptibility to attack by glucoamilase (97–98%) at 60 °C and 24 hours of saccharification, which suggested the absence of their resistance to the action of glucoamilase in the conditions of the experiment. The use of pullulanase in dextrinization of the analyzed starch, which was gelatinized and partly hydrolyzed by α-amylase (RS6.1%), enabled obtaining hydrolysates with the mass fraction of reducing substances in a range of 10–24% on DM basis with the process duration of 2 to 24 hours and the enzyme dose of 2–10 units, which contained mainly maltotriose, maltohexose and maltoheptose with their total amount of 45–60% on DM basis. The results indicate a need for further research of the biocatalytic action of pullulanase to develop new methods for enzymatic modification of starch.Использование деразветвляющих ферментов при гидролизе крахмала является актуальным направлением для получения новых видов крахмалопродуктов с контролируемыми свойствами и потенциалом для дальнейшего использования. Целью работы являлось изучение действия пуллуланазы (ЕС 3.2.1.41) на кукурузный амилопектиновый крахмал в нативном и клейстеризованном состоянии. Объектами исследований являлись амилопектиновый кукурузный крахмал и ферментный препарат Promozyme D6 (Novozymes, Дания). Для определения углеводного состава гидролизатов применяли метод высокоэффективной жидкостной хроматографии (ВЭЖХ), массовую долю редуцирующих веществ (РВ) определяли методом Лейна и Эйнона, для измерения динамической вязкости продуктов гидролиза крахмала был использован ротационный вискозиметр. Выявлено, что в нативном состоянии испытуемый крахмал проявил невысокую ферментативную восприимчивость к действию пуллуланазы с незначительными изменениями вязкости, растворимости и йодсвязующей способности образцов. Показано, что наибольшую активность на клейстеризованный крахмал пуллуланаза проявляла в первые 8 часов инкубации. Установлено, что максимальная степень гидролиза крахмала пуллуланазой через 8 часов при дозе 10 ед/г сухого вещества (СВ) составила 4,7% по СВ, йодсвязующая способность гидролизата D600 –0,343, при этом в контрольном опыте она составила D600 –0,154, а вязкость гидролизата снизилась с 7887 мПас · с до 4,3 мПа · с. Гидролизаты, охлажденные до 8 °C и выдержанные в течение 20 часов наряду с неохлажденными, проявили высокую атакуемость глюкоамилазой на 97–98% при 60 °C и 24 часа осахаривания, что указывало на отсутствие их резистентности к действию глюкоамилазы в условиях опыта. Использование пуллуланазы при декстринизации клейстеризованного и частично гидролизованного α-амилазой (РВ 6,1%) испытуемого крахмала позволяло получать гидролизаты с массовой долей редуцирующих веществ в пределах 10–24% по СВ при продолжительности процесса от 2 до 24 часов и дозировке фермента 2–10 ед., которые содержали в основном мальтотриозу, мальтогексозу и мальтогептозу с их суммарным количеством 45–60% по СВ. Результаты свидетельствуют о необходимости продолжения исследований биокаталитического действия пуллуланазы для разработки новых способов ферментативной модификации крахмала

Unresolved Multiple Stars and Galactic Clusters' Mass Estimates

If not properly accounted for, unresolved binary stars can induce a bias in the photometric determination of star cluster masses inferred from star counts and the luminosity function. A correction factor close to 1.15 (for a binary fraction of 0.35) was found in Borodina et al., which needs to be applied to blind photometric mass estimates. This value for the correction factor was found to be smaller than literature values. In an attempt to lift this discrepancy, in this work the focus is on higher order multiple stars with the goal of investigating the effect of triple and quadruple systems adopting the same methodology and data set as in the quoted work. The result is that when triple and quadruple, together with binary, systems are properly accounted for, the actual cluster mass (computed as all stars were single) should be incremented by a factor of 1.18-1.27, depending on the cluster and when the binary fraction α is 0.35. Fitting formulae are provided to derive the increment factor for different binary star percentages. © 2021. The American Astronomical Society. All rights reserved.

High precision measurement of the associated strangeness production in proton proton interactions

A new high precision measurement of the reaction pp -> pK+Lambda at a beam momentum of 2.95 GeV/c with more than 200,000 analyzed events allows a detailed analysis of differential observables and their inter-dependencies. Correlations of the angular distributions with momenta are examined. The invariant mass distributions are compared for different regions in the Dalitz plots. The cusp structure at the N Sigma threshold is described with the Flatt\'e formalism and its variation in the Dalitz plot is analyzed.Comment: accepted for publication in Eur. Phys. J.