PRODUCTION OF CYCLIC BETA-1,2-GLUCANS IN HCDC OF RECOMBINANT E. coli.

GUIDOLIN, L. SOLEDAD; CAILLAVA, ANA JOSEFINA; COUTO, ALICIA S.; CASABUONO, ADRIANA C.; COMERCI, DIEGO J.; ANDRÉS E. CIOCCHINI.

Mendoza

Congreso; LVIII Congreso Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología; 2022

SAIB

Bacterial polysaccharides are molecules with interesting biotechnological applications. Many bacterial carbohydrates have been purified from natural producers while others, particularly those where natural producers or isolation methods were undesirable, have been obtained by means of heterologous gene expression. Cyclic beta-1,2-glucans (CBG) are bacterial cyclic homopolysaccharides consisting in 17 up to 40 beta-1,2 linked glucoses. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that unsubstituted CBG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, making these molecules interesting targets for commercial/industrial development. Nowadays, there is no method to produce CBG by chemical synthesis. Furthermore, naturally synthesized CBG differ in the distribution of the degree of polymerization and in the presence and nature of chemical substituents in the final product, changing the properties of the molecule depending on the bacterial host. Finally, bacteria that synthesize CBG, mostly Rhizobiaceae and Brucellaceae members, are slow-growing or even pathogenic, which makes the scaling up of the process more difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand. Here, we present the production of CBG in a specially designed E. coli strain by means of the deletion of intrinsic polysaccharides biosynthetic genes and the heterologous expression of B. abortus enzymes involved in CBG synthesis. This strain synthesizes and secretes unsubstituted CBG with a degree of polymerization of 17 to 24 glucoses. Through high cell density culture (HCDC) in a 7L stirred tank bioreactor we obtained 6,9 g/L of crude product (total reducing sugars) in culture medium within 48 h culture. By Size Exchange Chromatography purification, we recovered 4,5 g CBG/L of culture (94 mgL-1h-1), the highest volumetric productivity reported to our knowledge. With this new approach, we are expanding the use of bacteria as a platform for the recombinant synthesis of polysaccharides such as the CBGs.