Metabolic channeling for biofuel production : Co-localization of Pdc and Adh

Abstract: Enhancing productivity in bioprocesses, especially for biofuel production, is crucial for achieving an environmentally and economically sustainable biotechnology industry.Metabolic channelling occurs in nature when the intermediate between two consecutive enzymes in a pathway is directed from the first enzyme to the second avoiding diffusion in the cytosol. This would be very advantageous in bioprocesses as it would increase efficiency of a particular pathway, reducing side products and protecting the cells from potential toxic intermediates. In recent years different strategies for emulating channelling effect wereproposed and used with very promising results. Clustering of enzymes seems to be the simplest way to create metabolic channelling. In this master thesis, four different strategies to co-localize enzymes in clusters are compared. The metabolic pathway chosen as a model was ethanol production by pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh). Chimeric proteins were genetically engineered and transformed in E. coli creating different strains. Ethanol production by the different strains was measured to compare production efficiency. Cell growth and protein expression were used for further understanding of the results. Strengths and weaknesses of each strategy and proposals for further improvement were discussed.