Перегляд за Автор "Klymenko, Natalia"

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Induction of auxins synthesis by Rhodococcus erythropolis IMV Ac-5017 with the addition of tryptophan to the cultivation medium
(2020) Pirog, Tatiana; Leonova, Natalia; Piatetska, Daria; Klymenko, Natalia; Zhdanyuk, Valentina; Shevchuk, Tetiana
The ability of surfactant producers to synthesize phytohormones expands the scope of their practical application and provides prospects for the development of microbial preparations with growth-stimulating properties. The possibility to intensify the phytohormone-stimulants synthesis by bacterial strains increases the efficiency of such preparations. Aim. The aim is to research the possibility of extracellular auxin synthesis induction in the presence of tryptophan in the cultivation medium of surfactant producer Rhodococcus erythropolis IMV Ac-5017 and establish the optimal concentration of tryptophan and time of introduction into the medium to ensure maximum synthesis of auxins. Methods. Biochemical, microbiological, biotechnological. Cultivation was performed in the liquid mineral medium using ethanol and waste sunflower oil as substrates. Tryptophan was added to the medium as a 1 % solution in an amount of 200 or 300 mg/l at the beginning of the cultivation process or at the end of the exponential growth phase. Phytohormones were isolated by triple extraction with organic solvents from the culture broth supernatant after surfactant extraction. Preliminary purification and concentration of phytohormones was performed by thin layer chromatography. Qualitative and quantitative determination of auxins was performed using high performance liquid chromatography. Results. It was found that regardless of the concentration and time of tryptophan introduction to the culture medium of R. erythropolis IMV Ac-5017 with both substrates, a significant increase (by two to three orders of magnitude) was observed in the amount of synthesized auxins compared to tryptophan-free medium. The highest concentration of auxins (5552–5634 μg/l) was achieved by adding 300 mg/l of tryptophan into the culture medium of R. erythropolis IMV Ac-5017 with ethanol, while without the precursor their amount was only 143 μg/l. In contrast to the cultivation of the strain on culture medium with ethanol, where the synthesis of auxins did not depend on the time of tryptophan introduction, R. erythropolis IMV Ac-5017 formed the maximum amount of auxins when 300 mg/l tryptophan was added to the culture medium with waste oil at the end of the exponential growth phase (2398 μg/l compared to 9.8 μg/l on the medium without tryptophan). As auxin compounds were identified: indole-3-acetic acid, indole-3-carboxylic acid and indole-3-butyric acid. However, the highest amount of indole-3-acetic acid was synthesized, the precursor of which is tryptophan. The synthesis of this auxin (the most common plant auxin) in the presence of 300 mg/l of tryptophan increased more than 40 times on ethanol medium and more than 700 times on medium with waste oil. Induction of auxin synthesis by strain R. erythropolis IMV Ac-5017 correlated with the activity of tryptophan transaminase: when cultured on ethanol without tryptophan, it was 138 nmol•min-1•mg-1 of protein, while cultured in the presence of precursor it was increased by 5.2 times (up to 714 nmol•min-1•mg-1 of protein). The obtained results suggest that indole-3-acetic acid biosynthesis by the strain IMV Ac-5017 occurs due to the formation of indole-3-pyruvate. Conclusions. Thus, it was established the possibility of increasing by two or three orders the amount of synthesized auxins in the case of low concentrations of tryptophan introducing to the culture medium of R. erythropolis IMV Ac-5017 not only with ethanol but also with industrial waste (waste oil). The obtained results can be considered as promising for use of exometabolites of R. erythropolis IMV Ac-5017 with growth-stimulating properties in crop production.
Influence of tryptophan on auxin-synthesizing ability of surfactant producer Acinetobacter calcoaceticus IMV B-7241
(2020) Pirog, Tatiana; Leonova, Natalia; Piatetska, Daria; Klymenko, Natalia; Shevchuk, Tetiana
Introduction. The aim of this work is to determine the optimal tryptophan concentration and the time of its addition into the culture medium of the surfactant producer Acinetobacter calcoaceticus IMV B-7241 to achieve maximum auxin synthesis. Materials and methods. Cultivation was carried out on a liquid nutrient mineral medium using as a substrate of ethanol and waste of biodiesel production (crude glycerol). Tryptophan was added into the medium as a 1% solution in an amount of 100, 200 or 300 mg/L at the beginning of the process or at the end of the exponential growth phase (48 h of cultivation). Phytohormones were isolated by three times extraction with organic solvents from the supernatant of the culture liquid after extraction of surfactants. The qualitative and quantitative determination of gibberellins was carried out by high performance liquid chromatography. Results and discussion. The results show that regardless of the time of addition of tryptophan in the culture medium of the strain IMV B-7241 with crude glycerol a significant increase in the synthesis of auxins compared with those on the medium without this precursor was observed. The highest concentration of auxins was achieved by adding 300 mg/L of tryptophan into the medium with both substrates. Thus, A. calcoaceticus IMV B-7241 synthesized 1404.73, 1295.04 and 4850.98 μg/L of auxins on the crude glycerol medium with 100, 200 and 300 mg/L tryptophan added at the end of exponential phase respectively (and without precursor the concentration of auxins was 175,4 μg/L). Increased synthesis of auxins by strain IMV B-7241 correlated with the activity of tryptophan transaminase (key enzyme of biosynthesis): under cultivation on crude glycerol without precursor it was 163 nmol•min-1•mg-1 of protein, while in the presence of 300 mg/L of tryptophan added at the end of the exponential growth phase the activity was in 3.2 times higher – 526 nmol•min-1•mg-1 of protein. The highest concentration of auxins under cultivation on ethanol was achieved when 300 mg/L of tryptophan were added at the beginning of cultivation – 2261.66 μg/L. Conclusion. The result of the work established the possibility of increasing by one or two orders of magnitude of synthesized auxins in the case of addition into the culture medium of A. calcoaceticus IMV B-7241 low concentrations of the precursor of their biosynthesis.
Ways of auxin biosynthesis in microorganisms
(2022) Pirog, Tatiana; Piatetska, Daria; Klymenko, Natalia; Iutynska, Galyna
Among plant hormones, auxins, in particular indole-3-acetic acid (IAA), are the most studied and researched. Almost all groups of soil microorganisms, both plant-associated and non-plant-associated bacteria, fungi, and phytopathogenic microorganisms are capable of producing auxins. Th e development of preparations for crop production is directly related to the production of bacterial strains with high auxin-synthesizing potential, which is possible only with a full understanding of the ways of regulation and synthesis of auxins in bacteria. Th e synthesis of auxins in microorganisms can take place in two ways: by the gradual conversion of tryptophan to IAA (tryptophan-dependent pathway) or by the use of other intermediates (tryptophan-independent pathway). Th e latter is poorly clarifi ed, and in the literature available today, there is only a small amount of information on the functioning of this pathway in microorganisms. Th e review presents literature data on the ways of auxin biosynthesis in diff erent groups of microorganisms, as well as approaches to the intensifi cation of indole-3-acetic acid synthesis. Th e formation of IAA from tryptophan can be carried out in the following ways: through indole-3-pyruvate, through indole-3-acetamide, and through indole-3-acetonitrile. Th e vast majority of available publications are related to the assimilation of tryptophan through the formation of indole-3-pyruvate as this pathway is the most common among microorganisms. Th us, it functions in rhizospheric, symbiotic, endophytic, and free-living bacteria. Th e concentration of synthesized IAA among natural strains is in the range from 260 to 1130 μg/mL. Microorganisms in which the indole-3-acetamide pathway functions are characterized by lower auxin-synthesizing ability compared to those that assimilate tryptophan through indole-3-pyruvate. Th ese include bacteria of the genera Streptomyces, Pseudomonas, and Bradyrhizobium and fungi of the genus Fusarium. The level of synthesis of IAA in such microorganisms is from 1.17•10−4 to 255.6 μg/mL. To date, only two strains that assimilate tryptophan via the indole-3-acetonitrile pathway and form up to 31.5 μg/mL IAA have been described in the available literature. To intensify the synthesis of indole-3-acetic acid, researchers use two main approaches: the fi rst consists in introducing into the culture medium of exogenous precursors of biosynthesis (usually tryptophan, less oft en indole-3-pyruvate, indole-3-acetamide, and indole-3-acetonitrile); the second — in increasing the expression of the corresponding genes and creating recombinant strains-supersynthetics of IAA. Th e largest number of publications is devoted to increasing the synthesis of IAA in the presence of biosynthesis precursors. Depending on the type of bacteria, the composition of the nutrient medium, and the amount of exogenously introduced precursor, the synthesis of the fi nal product was increased by 1.2—27 times compared to that before the intensifi cation. Th us, in the presence of 11 g/L tryptophan, Enterobacter sp. DMKU-RP206 synthesized 5.56 g/L, while in a medium without the precursor, it yielded only 0.45 g/L IAA. Recombinant strains Corynebacterium glutamicum ATCC 13032 and Escherichia coli MG165 formed 7.1 and 7.3 g/L IAA, respectively, when tryptophan (10 g/L) was added to the culture medium. Th e level of auxin synthesis in microorganisms may be increased under stress conditions (temperature, pH, biotic and abiotic stress factors), but in this case, the IAA concentration does not exceed 100 mg/L, and therefore this method of intensifi cation cannot compete with the others above.