OPTIMIZATION OF THE CULTIVATION CONDITIONS AND THE BASIC MOLECULAR TOOLS FOR ROSEOFLAVIN PRODUCER STREPTOMYCES DAVAWENSIS.

Roseoflavin (RoF), and its metabolic precursor 8-dimethylaminoriboflavin (AF), produced by Gram-positive bacteria Streptomyces davawensis and Streptomyces cinnabarinus, reveal a strong antibiotic effect against Staphyloccus aureus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus and other Gram-positive bacteria and could be promising for developing the new effective antibacterial drugs. In this work, we present the optimization of basic molecular tools for S. davawensis including total DNA preparation, PCR amplification of target gene, cell transformation. In addition, the compositions of cultivation media were studied and the optimal cultivation conditions for increased of RoF production have been developed.

[Construction of the flavinogenic yeast Candida famata strains with high riboflavin kinase activity using gene engineering].

The recombinant strains of the flavinogenic yeast Candida famata, which contain the DNA fragment consisting of the FMN1 gene (encoding the riboflavin kinase, enzyme that converts riboflavin to flavinmononucleotide) driven by the strong promoters (the regulated RIB1 or constitutive TEF1 promoter) were isolated. Riboflavin kinase activity in the isolated transformants was tested. The 6-8-fold increase of the riboflavin kinase activity was shown in the recombinant strains containing the integrated Debaryomyces hansenii FMN1 gene under the strong constitutive TEF1 promoter. The recombinant strains can be used for the following construction of flavinmononucleotide overproducers.

[Influence of manganese on iron accumulation and flavinogenesis in yeast Debaryomyces hansenii]. / Bplyv marhantsiu na akumuliatsiiu zaliza i flavinohenez u drizhdzhiv Debaryomyces hansenii.

In response to MnCl2 introduction the yeast Debaryomyces hansenii shows the increase of intracellular iron content and riboflavin biosynthesis level. Under iron deficiency the yeast cells sensitivity to manganese ions increases in spite of redundant synthesis of riboflavin. High concentration of iron in the culture results in the lower toxicity of Mn2+. Iron accumulation favours the cell resistance to manganese. Manganese causes the redundant synthesis of riboflavin as well as iron accumulation in streptonigrin-resistant mutants with a lowered iron content in cells. The Mn(2+)-resistant strains are phenotypically similar to the previously described Rib80(-)-mutants of D. hansenii with high iron content and riboflavin biosynthesis level. This suggests that riboflavin redundant synthesis and iron accumulation in the yeast cells are regulated by a common Mn(2+)-depending factor. A new method for isolation of the Rib80(-)-mutants is described. The method is based on the positive selection of manganese-resistant clones.

Oversynthesis of riboflavin by yeast Pichia guilliermondii in response to oxidative stress.

The effect of oxidative stress on riboflavin (vitamin B2) biosynthesis and iron accumulation in flavinogenic yeast P. guilliermondii was investigated. Treatment of P. guilliermondii cells with superoxidgenerating agent methylviologen leads to elevated production of malondialdyhyd (MDA) which reflects the overall cellular oxidation state. Increased iron content in the cells and enhanced productivity of flavinogenesis under these conditions has been shown too. Significant increasing of MDA and riboflavin production by yeast cells under iron deficiency was observed. Riboflavin overproducing P. guilliermondii mutant strains rib80, rib81 and hit, possess high iron transport and synthesize increased quantity of MDA. The role of riboflavin overproduction and activation of iron assimilation in the P. guilliermondii antioxidant defence is discussed.

[Riboflavin biosynthesis in yeast as a model for estimating mechanisms of regulating synthesis of biologically active substances]. / Biosyntez ryboflavinu u drizhdzhiv iak model' dlia doslidzhennia mekhanizmiv rehuliatsiï syntezu biolohichno aktyvnykh rechovyn.

General principles of regulation of transcription in eucaryotic cells are characterized. Investigations on riboflavin biosynthesis and regulation of this process in yeasts are summarized. The data obtained permit to consider riboflavin biosynthesis control as an interesting and convenient model capable to estimate the mechanisms various transcription factors action. These investigations are required in order to reveal general features of vitamins and relative compounds biosynthesis regulation.

[Ferrireductase from Pichia guilliermondii: properties and regulation of activity and synthesis]. / Ferireduktaza Pichia guilliermondii: vlastyvosti i rehuliarsiia aktyvnosti ta syntezu.

Properties of Pichia guilliermondii ferrireductase of the crude extracts and ammonium sulfate preparations were studied. NADH and Cu (II) ions are necessary for ferrireductase activity. Mg (II) also stimulates this reaction while oxygen acts as a slight inhibitor. Ferrireductase reduces Fe (III) in complex with citrate, iron-binding peptide from the culture medium of P. guilliermondii, rhodotorulic acid, coprogen, desferrioxamine B and EDTA. Mutants rib80, rib81 and hit of Pichia guilliermondii that have damaged system of riboflavin biosynthesis and iron transport regulation are characterized by high ferrireductase activity. Iron through negative feed-back mechanism regulates activity of ferrireductase synthesis in P. guilliermondii, Candida famata, Candida krusei, Candida boidinii, but not in Pichia pinus and Hansenula polymorpha.