[Physiological and biochemical characteristics of fungi of the genus Penicillium as producers of ergot alkaloids and quinocitrinins].

Four cultures of fungi of the genus Penicillium belonging to Furcatum Pitt subgenus, such as P. citrinum Thom, 1910; P. corylophilum Dierckx, 1901; P. fellutanum Biourge, 1923; and P. waksmanii Zaleski, 1927, produced the ergot alkaloids, namely, agroclavine-I, and epoxyagroclavine-I; their N-N-dimers, such as dimer of epoxyagroclavine-I and the mixed dimer of epoxyagroclavine-I and agroclavine-I; and also quinoline metabolites, namely, quinocitrinin A and quinocitrinin B. Physiological and biochemical characteristics of the producers were studied. Optimal conditions for the biosynthesis of metabolome components were determined. Zinc additive to the medium stimulated the biosynthesis of the ergot alkaloids in all cases; citrinin production was increased only in P. citrinum, and that was suppressed in P. corylophinum, P. fellutanum, and P. waksmanii. This testifies that genes of the biosynthesis pathways are located in the different clusters of the producers.

New approaches to identification and activity estimation of glyphosate degradation enzymes.

We propose a new set of approaches, which allow identifying the primary enzymes of glyphosate (N-phosphonomethyl-glycine) attack, measuring their activities, and quantitative analysis of glyphosate degradation in vivo and in vitro. Using the developed approach we show that glyphosate degradation can follow different pathways depending on physiological characteristics of metabolizing strains: in Ochrobactrum anthropi GPK3 the initial cleavage reaction is catalyzed by glyphosate-oxidoreductase with the formation of aminomethylphosphonic acid and glyoxylate, whereas Achromobacter sp. MPS12 utilize C-P lyase, forming sarcosine. The proposed methodology has several advantages as compared to others described in the literature.

[The influence of medium composition on alkaloid biosynthesis by Penicillium citrinum].

The fungus P. citrinum produces secondary metabolites, clavine ergot alkaloids (EA), and quinoline alkaloids quinocitrinines (QA) in medium with various carbon and nitrogen sources and in the presence of iron, copper, and zinc additives. Mannitol and sucrose are most favorable for EA biosynthesis and mannitol is most favorable for QA. Maximum alkaloid production is observed on urea. Iron and copper additives in the medium containing zinc ions stimulated fungal growth but inhibited alkaloid biosynthesis. The production of these secondary metabolites does not depend on the physiological state of culture, probably due to the constitutive nature of the enzymes involved in biosynthesis of these substances.

[Thiomorpholine transformation by the fungus Bjerkandera adusta].

A screening of lignin-degrading basidial fungi that can grow in the presence of thiomorpholine derivatives (the mixture of 1,4-perhydrothiazines) has been performed. Strain Bjerkandera adusta VKM F-3477 was shown to have the maximal rate of growth in the presence of these compounds, and its capacity for thiomorpholine degradation was studied. The methods of quantitative analysis of thiomorpholine and its degradation products on the basis of thin layer chromatography and high-performance liquid chromatography were developed. It was shown that the B. adusta strain did not utilize thiomorpholine as a carbon source but transformed it into thiomorpholine sulfoxide that accumulated in the medium. Mn peroxidase produced by B. adusta in the course of thiomorpholine transformation is not directly involved in its oxidation.

[Biodegradation of phenanthrene by Pseudomonas bacteria bearing rhizospheric plasmids in model plant-microbial associations]. / Biodegradatsiia fenantrena rizosfernymi plazmidosoderzhashchimi bakteriami roda Pseudomonas v model'nykh rastitel'no-mikrobnykh assotsiatsiiakh.

The consumption phenanthrene in soil by model plant-microbial associations including natural and transconjugant plasmid-bearing rhizospheric strains of Pseudomonas fluorescens and P. aureofaciens degrading polycyclic aromatic hydrocarbons was studied. It was shown that phytoremediation of soil polluted with phenanthrene in the rhizosphere of barley (Hordeum sativum L.) was inefficient with the absence of the degrading strains. Inoculation of barley seeds with both natural and transconjugant plasmid-bearing Pseudomonas strains able to degrade polycyclic aromatic hydrocarbons (PAH) protected plants from the phytotoxic action of phenanthrene and favored its degradation in soil. Rape (Brassica napus L.) was shown to be an appropriate sentinel plant, sensitive to phenanthrene, which can be used for testing the efficiency of phenanthrene degradation in soil. The biological test with the use of sensitive rape plants can be applied for estimation of the efficiency of phyto/bioremediation of PAH-polluted soils.

[The alkaloids of Penicillium aurantiogriseum Dierckx (1901) var. aurantiogriseum VKM F-1298]. / Alkaloidy griba Penicillium aurantiogriseum Dierckx (1901) var. aurantiogriseum BKM F-1298.

The fungus Penicillium aurantiogriseum var. aurantiogriseum VKM F-1298 produces two benzodiazepine alkaloids (anacine and aurantine) and one diketopiperazine alkaloid (aurantiamine). When cultured in a submerged mode in Abe medium, the alkaloids are mostly secreted into the medium. The dynamics of aurantine and aurantiamine accumulation in the medium is characterized by the presence of a relatively sharp maximum in the idiophase, whereas the accumulation of anacine in the medium is characterized by an extended plateau and occurs concurrently with fungal growth.

[Reversed-phase high performance liquid chromatography of the products of microbiological degradation of toluene ]. / Analiz produktov mikrobiologicheskoi degradatsii toluola metodom obrashchenno-faznoi zhidkostnoi khromatografii.

Conditions have been selected for a reversed-phase high-performance liquid chromatographic assay of intermediate products formed in the course of utilization of toluene by Pseudomonas putida. The composition of products indicates that degradation of toluene by strain BS590-P proceeds primarily through the formation of benzoate and catechol. This is followed by degradation of catechol via ortho-cleavage. In strain BS3701-P, toluene oxidation involves both the side chain and the aromatic ring.

[Optimization of conditions for storage and cultivation of the fungus Claviceps sp.--a producer of the ergot alkaloid agroclavine]. / Optimizatsiia uslovii khraneniia i kul'tivirovaniia griba Claviceps sp.--produtsenta érgoalkaloida agroklavina.

Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain s 106 were studied. The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90-95%). Storage of the culture at -70 degrees C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.

[Analysis of secondary metabolites of microscopic fungi of the genus Penicillium by chromatographic techniques]. / Analiz vtorichnykh metabolitov mikroskopicheskikh gribov roda Penicillium khromatograficheskimi metodami.

Combinations of various regimens of thin-layer chromatography and high-performance liquid chromatography (HPLC) was efficient in analyzing 39 nitrogen-containing secondary metabolites (alkaloids) produced by 12 strains of microscopic fungi of the genus Penicillium. Chromatographic mobility of alkaloids on Silufol plates was determined in the following systems (following staining with the Ehrlich reagent): (a) chloroform, methanol, and 25% NaOH (90:10:1, 90:10:0.1, or 80:20:0.2); (b) chloroform and acetone (9:1); and (c) ethyl acetate, methanol, and 25% NH4OH (85:15:10). Conditions for separation of clavine alkaloids by HPLC on Spherisorb ODS2 and Supelcosil LC-18 columns (gradient elution) were optimized. Retention values of 22 alkaloids were compared to those of agroclavine and roquefortine.

[Optimization of the medium and cultivation conditions of Penicillium roquefortii f39 producing the diketopiperazine alkaloid roquefortine]. / Optimizatsiia sredy i uslovii kul'tivirovaniia Penicillium roquefortii f39--produtsenta diketopiperazinovogo alkaloida rokefortina.

We optimized the medium for cultivation of Penicillium roquefortii f39, a producer of roquefortine. In this medium, the roquefortine yield increased 1.5-2-fold. The increase in roquefortine content was associated with high biomass yield, but not with an increase in biosynthetic activity of the mycelium. Direct correlation was found between extracellular roquefortine concentration and amount of the inoculum. The introduction of sucrose into the growth medium allowed us to increase the concentration of roquefortine during fermentation to 90 mg/l.

[Selection of ergot alkaloid producers by induced mutagenesis]. / Poluchenie produtsentov érgoalkaloidov metodom indutsirovannogo mutageneza.

Using the induced mutagenesis technique, A series of genetically modified Claviceps sp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.

[Factors contributing to roquefortine yield variability during cultivation of penicillium roquefortii]. / Prichiny nestabil'nogo vyshoda rokefortina pri kul'tivirovanii Penicillium roquefortii.

Variability in the roquefortine yield was shown to be associated with its consumption by the mycelium during isolation of the end product, which depended on temperature, time of culture liquid storage, and biomass concentration. This was also related to the presence in chloroform of chlorocarbonic acid ethyl ester that reacted with roquefortine.

[Minor alkaloids from the fungus Penicillium roquefortii Thom 1906]. / Minornye alkaloidy gribov Penicillum roquefortii Thom 1906.

New spatial of clavine alkaloids, distinguished by low chromatographical mobility, have been isolated from the collection and mutant strain of Penicillium roquefortii, in addition to alkaloids roquefortine, 3,12-dihydroroquefortine, isofumigaclavines A and B, festuclavine, and chanoclavine-I, characteristic of this fungal species. In has been shown that the collection strain produces isomers of agroclavine and epoxyagroclavine, and the mutant strain produces isomers of fumigaclavines A and B, festuclavine, and chanoclavine.

Involvement of naphthalene dioxygenase in indole-3-acetic acid biosynthesis by Pseudomonas putida.

Two variants of plant growth-promoting strain Pseudomonas putida BS1380 harboring the naphthalene degradative plasmid pBS2 and the recombinant plasmid pNAU64 that contains the genes encoding for naphthalene dioxygenase were constructed by conjugation. The ability of this strain to produce phytohormone indole-3-acetic acid from different carbon sources was studied. Indole-3-acetic acid synthesis by these transconjugants was 15-30 times as much in contrast to a wild-type strain with glucose as the sole carbon source. No difference was observed in other carbon or nitrogen sources. It is suggested that naphthalene dioxygenase is involved in the conversion of indole-3-pyruvic acid to indole-3-acetic acid.

[Effect of the mutation process on formation of alkaloids in Penicillium roquefortii BKM F-141 and P. fellutanum BKM F-1073]. / Vliianie mutatsionnogo protsessa na obrazovanie alkaloidov u Penicillium roquefortii BKM F-141 i P. fellutanum BKM F-1073.

Mutant strains of Penicillium roquefortii VKM F-141 and P. Fellutanum VKM F-1073 were obtained by mutagenesis induced by ultraviolet irradiation, N-methyl-N-nitronitrosoguanidine and bromouracil. By the rates of alkaloid production, the mutant strains can be divided into three groups: 1) unable to synthesize alkaloids; 2) with a high rate of biosynthesis; 3) with changed alkaloid composition. Compounds not characteristic of wild-type cultures were found in alkaloid fractions of some mutant strains.