In this review, we discuss the progress in the study and modif ication of subtilisin proteases. Despite longstanding applications of microbial proteases and a large number of research papers, the search for new protease genes, the construction of producer strains, and the development of methods for their practical application are still relevant and important, judging by the number of citations of the research articles on proteases and their microbial producers. This enzyme class represents the largest share of the industrial production of proteins worldwide. This situation can explain the high level of interest in these enzymes and points to the high importance of designing domestic technologies for their manufacture. The review covers subtilisin classif ication, the history of their discovery, and subsequent research on the optimization of their properties. An overview of the classes of subtilisin proteases and related enzymes is provided too. There is a discussion about the problems with the search for (and selection of) subtilases from natural strains of various microorganisms, approaches to (and specif ics of) their modif ication, as well as the relevant genetic engineering techniques. Details are provided on the methods for expression optimization of industrial subtilases of various strains: the details of the most important parameters of cultivation, i. e., composition of the media, culture duration, and the inf luence of temperature and pH. Also presented are the results of the latest studies on cultivation techniques: submerged and solid-state fermentation. From the literature data reviewed, we can conclude that native enzymes (i. e., those obtained from natural sources) currently hardly have any practical applications because of the decisive advantages of the enzymes modif ied by genetic engineering and having better properties: e. g., thermal stability, general resistance to detergents and specif ic resistance to various oxidants, high activity in various temperature ranges, independence from metal ions, and stability in the absence of calcium. The vast majority of subtilisin proteases are expressed in producer strains belonging to different species of the genus Bacillus. Meanwhile, there is an effort to adapt the expression of these enzymes to other microbes, in particular species of the yeast Pichia pastoris.

Methylotrophic yeasts have been used as the platform for expression of heterologous proteins since the  1980’s. They are highly productive and allow producing eukaryotic proteins with an acceptable glycosylation level.  The first Pichia pastoris-based system for expression of recombinant protein was developed on the basis of the treeexudate-derived strain obtained in the US southwest. Being distributed free of charge for scientific purposes, this system has become popular around the world. As methylotrophic yeasts were classified in accordance with biomolecular  markers, strains used for production of recombinant protein were reclassified as Komagataella phaffii. Although patent  legislation suggests free access to these yeasts, they have been distributed on a contract basis. Whereas their status  for commercial use is undetermined, the search for alternative stains for expression of recombinant protein continues.  Strains of other species of methylotrophic yeasts have been adapted, among which the genus Ogataearepresentatives prevail. Despite the phylogenetic gap between the genus Ogataeaand the genus Komagataellarepresentatives, it turned out possible to use classic vectors and promoters for expression of recombinant protein in all cases. There  exist expression systems based on other strains of the genus Komagataellaas well as the genus Candida. The potential  of these microorganisms for genetic engineering is far from exhausted. Both improvement of existing expression systems and development of new ones on the basis of strains obtained from nature are advantageous. Historically, strains  obtained on the southwest of the USA were used as expression systems up to 2009. Currently, expression systems  based on strains obtained in Thailand are gaining popularity. Since this group of microorganisms is widely represented  around the world both in nature and in urban environments, it may reasonably be expected that new expression systems for recombinant proteins based on strains obtained in other regions of the globe will appear.