Study of the synthesis and degradation of the carbon reserve polymer in Euglena gracilis

MUCHUT, ROBERTINO J.; CALLONI, RODRIGO D.; ARIAS, DIEGO G.; IGLESIAS, ALBERTO A.; GUERRERO, SERGIO A.

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Euglena gracilis is a fresh water protist able to grow photosynthetically orheterotrophically using a diverse array of organic compounds as the solesource of carbon and energy for growth. E. gracilis is a suitable source forthe generation of several compounds used for the production ofcosmeceuticals, nutraceuticals, food, paramylon and wax esters. When grownaerobically it produces an insoluble β-1,3-glucan as storage polymer.Synthesis of carbon reserve polymer (paramylon) occurs via UDP-glucose, akey metabolite of the carbohydrate pathways present in most organisms,generated in a reaction catalyzed by UDP-glucose pyrophosphorylase (EC2.7.7.9; UDP-GlcPPase).The enzyme from E. gracilis was expressed inEscherichia coli cells. The purified enzyme was kinetically characterized,catalyzing UDP-glucose synthesis with a Vmax value of 3350 U/mg, and affinityfor substrates of 0.24 mM and 0.17 mM for glucose-1P and UTP respectively.We have also performed, for the first time, confocal microscopy assays tovisualize the intracellular location of UDP-GlcPPase.On the other hand, degradation of paramylon is catalyzed by β-1,3-glucanases. We found several transcripts corresponding to endo-β-1,3-glucanase (EC 3.2.1.39) belonging to different glycoside hydrolases (GH)families. Activity β-1,3-glucanase were measured achieving 0.05 U/mg fromsoluble fractions of crude extract and 0.03 U/mg from membrane fractionssolubilized with detergent. In order to deepen the knowledge in thedegradation of the paramylon, we performed the novo synthesis andmolecular cloning of two genes coding for two endo-β-1,3-glucanase, onefrom GH 17 family and the other one from GH 64 family. These genes wereused for recombinant expression in Escherichia coli cells. These resultsobtained allow us to improve our understanding about carbon reservemetabolism in Euglena gracilis for biotechnological purpose.