A GLYCOENGEENERING PLATAFORM FOR DESIGN AND HIGH YIELD PRODUCTION OF RECOMBINANT NEUTRAL CYCLIC BETA GLUCANS

CAILLAVA, AJ; COMERCI DJ; GUIDOLIN LS; CASABUONO, AC; COUTO, AS; CIOCCHINI AE

Congreso; LVII Reunión Anual de SAIB y el XVI Congreso Anual de la Asociación Civil de Microbiología General (SAMIGE); 2021

SAIB-SAMIGE

Cyclic beta-1,2-glucans (CBG) are ring-shaped bacterial polysaccharides produced by Rhizobiaceae and Brucellaceae members. They are synthesized as a mixture of molecules with different ring sizes that vary from 17 to more than 28 glucoseunits depending on the bacterial species. CBG can be neutral or decorated with different molecules that confers a negative charge and the amount and identity of the substituent depends on the producer strain. CBG have a particular structure with a hydrophilic surface and a hydrophobic cavity that can include poorly soluble molecules. Due to these qualities, they can be used for drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators. There is no method to produce CBG by chemical synthesis. In addition, the bacteria that synthesize them naturally are slow-growing or even pathogenic, which complicates and makes the scaling process more expensive, as well as the fact that CBG must be purified from complex mixtures with other bacterial polysaccharides. All these disadvantages prevent the high-scale production of CBG, hampering potential industrial applications. To circumvent these problems, we developed a technological platform for the standardized production of ?recombinant CBG?. This platform is based on the expression of different engineered enzymes involved in CBG synthesis in a specially designed E. coli strain. Here, we present the production of unsubstituted (neutral) CBG. We first developed and optimized the strain and established the culture conditions to produce and secrete CBG. After that, we performed the production of CBG in an Erlenmeyer scale and calculated the stoichiometric parameters. The produced polysaccharides were characterized by TLC and mass spectrometry (MALDITOF) verifying that they were unsubstituted with a degree of polymerization of 17 to 25 glucose units. Finally, we scaled up recombinant neutral CBG production in high cell density culture (HCDC) conditions using a laboratory-scale bioreactor achieving 4.4 g/L of CBG in the culture supernatant and 6.1 g/L of cell associated CBG. This is the highest yield and volumetric productivity of CBG reported so far.