Bet on a horse
Biologists tame microorganisms for targeted drug delivery
Andrey Subbotin, "Search"
Cancer, diabetes, obesity, neurodegenerative diseases that overtake people in old age, inflammatory processes – this is an incomplete list of unresolved medical problems faced by modern man. In the treatment of such diseases, one of the key places is occupied by pharmaceuticals based on isoprenoid compounds of the steroid series. Steroids are very common in nature, as they perform the most important functions in all living things. Microorganisms are capable of destroying steroid molecules or modifying their structure. Today, it is the microbiological transformation of phytosterols (mixtures of sterols of plant origin) that is the technological basis for the production of synthons, from which pharmaceutical preparations such as adrenal cortex hormones, progestins, mineralocorticoids, sex hormones and non-hormonal steroids are then produced. They are used in veterinary medicine, food industry, agriculture. According to statistics, the total needs of the world pharmaceutical industry in steroid substances exceed 1,500 tons per year. The market of medicines produced from them is second only to antibiotics.
The discovery of new promising molecules, including those with antiviral and neuroprotective effects, the growing demand for vitamin D, as well as the reorientation of the production schemes of drugs for gastroenterology (such as bile acids) to the use of phytosterols as primary raw materials, allow us to predict a significant increase in the demand of the world market for steroid synthons. Microorganisms are already used for processing phytosterols in industrial volumes in a number of countries (USA, Germany, India, China, Spain, etc.). Russia, having huge resources in the form of sterine-containing waste from the forest chemical complex, does not yet have the production of a full cycle of steroid pharmaceutical substances, including vital medicines. Therefore, the needs of domestic healthcare are covered by imported supplies. Meanwhile, only 6 of the largest pulp and paper mills in our country produce over 300 thousand tons of tall products - sterine–containing raw materials, which are mostly simply burned, harming the environment.
Genetic technologies can provide a solution to these problems. The Russian Science Foundation is funding the project "Metabolic engineering of microbial producers for Industrial Biotechnology: obtaining valuable isoprenoid synthons from natural sterols". At the request of the Search, Marina DONOVA, the project manager, Doctor of Biological Sciences, chief researcher at the G.K. Scriabin Institute of Biochemistry and Physiology of Microorganisms of the Pushchinsky Scientific Center for Biological Research of the Russian Academy of Sciences, told about how the work is being carried out under this grant.
– For the industrial production of synthons from phytosterols, natural strains of bacteria that are not dangerous to humans and animals are used today, – Marina Viktorovna informs, introducing the case. – However, the problems associated with the low productivity of the strains used, incomplete conversion of phytosterols and the formation of by-products complicating the purification of target steroids have not been solved. Even today, it is important to expand the lines of target products with high added value obtained directly from phytosterols. This will help to reduce the production schemes of final pharmaceutical ingredients, replace multi-stage chemical syntheses with single-stage biotechnologies, increase the profitability of production and reduce environmental risks. Actually, for this purpose we study the mechanisms of functioning and regulation of metabolic pathways of sterol oxidation in actinobacteria. The goal is to create a new generation of industrial cellular biocatalysts. Thanks to them, it is possible not only to improve the properties of microbial strains by suppressing undesirable enzymatic activities, but also to redirect metabolic flows in the cell towards the formation of valuable steroid and isoprenoid compounds. Controlled expression of foreign steroidogenesis systems in microbial hosts opens up new ways.
– To express is to intensify?
– This is learning how to transform the hereditary information of a gene into a functional product in bacteria or yeast. That is, to get a connection that has certain specified properties.
– What kind of bacteria are you working with?
– The right question. It is important to choose the "chassis", the working platform of the microorganism, on the basis of which these new microbial cell factories will be created. Unfortunately, such workhorses traditionally used by geneticists as Escherichia coli bacteria, for which a solid genetic toolkit has been created, are of little use for these purposes. And the difficulty of working with steroids is that they are practically insoluble in water, – says Marina Viktorovna. – For example, the solubility of cholesterol does not exceed 10 mg per liter, and industrial catalysts must effectively convert steroids in concentrations exceeding their solubility by tens of thousands of times. In addition, it is necessary that the steroid substrate effectively penetrates into the microbial cell, for which it requires a hydrophobic surface and also effective transport systems for such compounds. We strive to learn more about the genome, own genes of steroids, regulation of their transcription, the metabolome of the microbial host organism, about the features of cascades of reactions of degradation of steroids and enzymes, - Marina Viktorovna delves into details, – therefore Mycobacterium Mycolicibacterium smegmatis are chosen as the workhorse in our project. Previously, for such microorganisms, we developed genetic tools for creating mutant strains with blocks at certain stages of the catabolic pathway of sterol degradation.
– How is the research process structured?
– We are conducting it in several directions. Firstly, we are trying to switch the oxidative degradation streams of the sterols side chain in mycolicybacteria cells to the accumulation of the products of its incomplete oxidation that we need.
– This is one direction...
– And the second is the creation of transgenic microorganisms that produce certain hydroxysteroids. Their biological activity depends on the degree of oxidation of the molecule and the presence of various functional substituents. We plan to obtain transgenic strains of mycobacteria producing hydroxysteroids from natural sterols based on a unique bacillary mutant protein. This approach is promising both for the bio-production of highly demanded hydroxylated steroids from phytosterol, and for world-class scientific research. In the future, this methodology may become the basis for the creation of other microbial biologically active compounds of various therapeutic purposes.
In addition, the grant works, which will last until 2024, provide for the implementation of a method for obtaining genetically engineered strains capable of selectively hydroxylating steroids using fungal enzymes.
– Unlike bacteria, fungi, as a rule, cannot completely destroy steroid molecules. Undesirable steroid compounds are formed for us, which significantly complicates the purification of the resulting products," explains Marina Donova. – But in the course of earlier studies of three hundred strains of various mycelial fungi, it was possible to identify several strains with unique properties. This allowed not only to confirm the functions of genes, but also to obtain recombinant strains capable of selective production of specified hydroxysteroids. We will also try to create strains producing new steroids with antiviral activity, as well as steroids effective against human papillomavirus, rotavirus, rhinovirus, without cytotoxic effects. Recently, high inhibitory activity of such steroids against coronaviruses SARS-CoV-2 and HCoV-OC43 has been shown in vitro.
In general, as a result of the implementation of this RNF grant, we expect not only to obtain new scientific data on the functioning of heterologous P450 monooxygenases in microbial hosts, but also to expand methodological approaches and create promising recombinant strains for practice. Research is a priority, and the implementation of the ideas laid down in the project can contribute to the creation of innovative biotechnologies," Marina Viktorovna emphasized. – The results are important not only for biotechnological applications, but also for biomedical research, as they open up prospects for identifying new targets for the creation of targeted drugs.
– For research of this scale, modern equipment and well-trained personnel are needed.
– We're fine with that. The team has specialists in the field of microbiology, genetic engineering, bioinformatics, chemistry, biotechnology. Moreover, with the experience of creating industrial microbial producers, developing full-cycle biotechnologies for the production of pharmaceutical substances and steroid intermediates, they have already carried out major Russian and international projects in the field of industrial microbiology and synthetic biology. More than half of the employees are young scientists. This laboratory structure allows us not only to create new strains, but also to develop technologies from the idea to the final crystalline product.
– The project is being conducted not only with the money of the RNF. Who else supports your work?
– The co-financing of the project is carried out by the Biochemical holding "Orghim". It is one of the world leaders in the field of production of "green chemistry" products, purposefully engaged in the implementation of scientific developments in industry. The companies of the holding develop environmentally friendly products of "green chemistry", including promising ones, such as phytosterol. Orghim invests in programs for the development of the forest chemical complex, its plans also include the implementation of projects to create new technologies and preparations from phytosterols.
– What are your plans for the future? Are you already thinking about the next steps?
– In the near future, of course, we plan to carry out this project and create a solid foundation for future research. We are very grateful to the RNF for its support. The grant funds helped us to attract a number of additional specialists and purchase the necessary equipment and consumables. And we have a lot of interesting ideas for the future, we would have enough strength and opportunities to implement them.
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