Gerardo Huerta-Beristain1, Guillermo Gosset2, and Alfredo Martínez2. (1) Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, UNAM, Av. Universidad No. 2001, Cuernavaca, 62210, Mexico, (2) Ingeniería Celular y Biocátalisis, Instituto de Biotecnología, UNAM, Av. Universidad 2001. Col. Chamilpa, Cuernavaca, 62210, Mexico
Ethanologenic Escherichia coli KO11 was engineered to channel glucose trough the Entner-Doudoroff (ED) and pentose phosphate (PP) pathways using a phosphoglucose isomerase interruption into the glycolytic pathway. KO11 pgi- was evolved to recover the capacity to grow in mineral media with 4% glucose under anaerobic conditions, and a homoethanologenic derivative was obtained deleting the pta, ack and ldh genes, obtaining KO11 PPAL-. Main results show that activity values for glucose-6-phosphate dehydrogenase (ZWF) and the Entner-Doudoroff (ED) pathway enzymes increased 17-fold and 2-fold (respectively) in KO11 PPAL- in comparison with KO11. Increased expression of pyruvate decarboxylase and alcohol dehydrogenase in KO11 PPAL- allowed to obtain specific ethanol formation rates similar to those found in KO11, but with half the cell mass and without carbon flux to acetate, formate and lactate, i.e. a large ethanol/glucose yield in mineral media. These results demonstrate that it is possible to obtain the same carbon flux using the PP and the ED pathways as alternative routes for the Embden-Meyerhoff-Parnas pathway for the glucose catabolism.
