Genetic engineering of cyclomaltodextrin glucanotransferases

Studies of cyclic oligosaccharides from six, seven and eight glucose residues, designated as alpha-, beta- and gamma-cyclodextrins, respectively, and everything related to them have been going on for 130 years. In this review, the history of the study of these molecules is briefly considered. The interest in cyclodextrins is caused by their ability to form inclusion complexes with a number of organic and inorganic compounds, radically changing some of their properties. This is widely used in the pharmaceutical, cosmetic and food industries, and beta-cyclodextrin is even registered as a food additive E459. Cyclodextrins are obtained from starch under the action of cyclodextringlucanotransferase (CGTase) enzymes, a characteristic feature of which is their non-strict specificity in relation to the types of oligosaccharides produced. The main producers of these enzymes are a group of bacteria of the order Bacillales, which unites several families (Paenibacillaceae, Bacillaceae, Thermoactynomicetaceae, etc.), but in last years CGTases have been found in a wide range of bacteria and archaea. The genetic engineering of CGTases began in the middle of 1980s, after the CGTase gene from Paenibacillus macerans (formerly Bacillus macerans) was cloned and sequenced for the first time, and during this period rather noticeable progress was made in understanding the organization and functioning of these enzymes, including using X-ray diffraction analysis. With the help of site-directed mutagenesis, error-prone PCR, as well as by creating chimeric forms of these enzymes, certain successes have been achieved in recent decades in changing (improving) the specificity of their action. Suitable leader peptides are used to increase the synthesis and secretion of genetically engineered CGTases, and various heterologous producers are also proposed, including the bacteria Escherichia coli, B.subtilis, Lactococcus lactis and the methylotrophic yeast Koagataella phaffii.