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Autor
Calderón Pérez-Lozao, Claudia
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Abstract
Glycolipids are a structurally diverse group of macromolecules that are present in almost all living organisms. Thanks to their physicochemical properties, they open a huge range of applications, among which their use for the development of new therapies in the field of biomedicine stands out. Within this group, the glycosphingolipid α-galactosylceramide has been shown to have potent antitumor activity in murine models and in vitro models.
Since their findings, such activity has been attributed to their ability to activate the iNKT cell subpopulation. As a consequence of this activation, cellular stimulation occurs, generating a response within the immune system involved in various defense mechanisms that are related to numerous infectious, autoimmune and oncogenic diseases.
From this molecule, the potent synthetic analog, KRN7000, was arrived at, which has been shown to be the prototype antigen of iNKT cells. Subsequently, multiple KRN7000 analogs have been developed that contain a range of different biochemical modifications with the goal of more accurately controlling the quality of cellular responses resulting in a long list of immunotherapeutic protocols with different applications.
Given its potential uses, the biotechnological production of KRN7000 is proposed as an alternative to its chemical synthesis in order to develop a more sustainable production process. For this, it is necessary to carry out the expression of the recently characterized enzyme, α -Galactosyltransferase, responsible for catalyzing the α-glycosidic bond that makes up the molecule. And, on the other hand, it is necessary to develop through metabolic engineering a biological platform in which it is possible to increase the availability of the ceramide lipid acceptor where the synthesis reaction will take place.
In this project, the design of the biological platform is approached by selecting as host the yeast Saccharomyces cerevisiae, through the overexpression of two genes involved in the synthesis pathway of complex sphingolipids. The different strategies used for the generation of the expression vectors are exposed and an alternative of a cloning method is proposed to arrive at the vectors that allow the overexpression of said genes. In the same way, the cloning design of the α-Galactosyltransferase enzyme is addressed, ready to be executed. Additionally, a detailed vision is given on the different points of the KRN7000 that are susceptible to being modified, as well as their applications.
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