ИСТИНА

Цель проекта: тестирование возможности использования белков-транспортеров гидрофобных соединений для повышения 1) эффективности поглощения холестерина клетками микроорганизмов и, вследствие этого, 2) эффективности его биотрансформации локализованной в клетках стероидогенной Р450scc-системой.

The use of transgenic microorganisms for the synthesis of steroid compounds is quite popular. Most of the currently available industrial strains have been improved through traditional technologies based on mutagenesis and selection. However, to obtain microorganisms that produce certain steroids with a high yield, it is often necessary to solve tasks related to fundamental scientific problems (regulation of gene expression, change in protein topogenesis, change in the direction of metabolic pathways in the cell, etc.). Developing the novel approaches used for metabolic engineering (promoters, terminators, episomal and integrative expression systems, CRISPR/Cas9 technology for rapid strain modification, etc.), the most important studies in the field of biotechnology of steroids are carried out using recombinant DNA technologies. For example, strains of microorganisms with reconstructed enzymatic P450 systems that synthesize steroid hormones in the human body were obtained. Due to the fact, that the steroid substrates of these P450 cytochromes are extremely hydrophobic, which limits their absorption by the cell, such strains demonstrate a low yield of the target product. To increase the solubility of steroids, organic solvents or detergents are usually used which often affect cell membranes, disrupting the integrity and viability of cells and, thus, reducing their biocatalytic activity. Within the framework of the project it is proposed to use a new approach to increase the efficiency of the steroid substrate delivery into the cell - the inclusion of the active carrier protein of steroid compounds in the cell membrane. The project goal: testing the possibility of using membrane protein transporters of hydrophobic compounds to increase 1) the efficiency of cholesterol absorption by the cells of microorganism and, therefore, 2) the effectiveness of its biotransformation by the steroidogenic P450scc system localized in the cells. This approach was not been used previously in world practice. The following transporter proteins will be used as agents for cholesterol delivery: in the case of bacteria, a deleted version of the StAR protein, a natural partner of cytochrome P450scc, which delivers cholesterol to the mitochondria of the adrenal cortex; in the case of yeast, an ABC1 transporter capable of transporting hydrophobic compounds into yeast cells. The use of heterologous models (bacteria E. coli and yeasts Y. lipolityca expressing cDNAs encoding proteins of the bovine adrenal cortex P450scc system (cytochrome P450scc (CYP11A1), adrenodoxine, and adrenodoxin reductase) and the same strains additionally expressing cDNA of transporter proteins) will allow us to evaluate the effects of StAR and ABC1 transporter on the efficiency of biotransformation of cholesterol to pregnenolone in target strains and, therefore, the possibility of applying this approach for optimization of the functioning of P450scc system in cells of prokaryotic and eukaryotic microorganisms. The need for steroid drugs is increasing every year, and studies aimed at constructing new “microbial cell factories” - productive and viable microorganisms that carry out environmentally friendly bioprocesses with the aim of obtaining specific steroid drugs, are still of current interest. In case of success, the new approach that will be tested within the project, will expand the arsenal of methods that can be used to create strains for the synthesis of widely sought-after steroid compounds.