Artificial Cellulosomes and Arsenic Cleanup: From Single Cell Programming to Synthetic Yeast Consortium.

摘要

As our society marches toward a more technologically-inclined and industrialized future, energy and environmental sustainability are two of the most challenging problems we face today. With the aid of recent advances in recombinant molecular technology, metabolic engineering has been employed on a variety of host organisms to improve biosorption and biocatalytic capabilities. This has shown immense promise and has become an attractive tool for bioremediation and biofuel production. In regards to these challenges, this dissertation focuses on the use of metabolic engineering for biofuel production and arsenic remediation.;The first objective of this dissertation was to create an efficient and inexpensive whole-cell biocatalyst in an effort to produce economically compatible and sustainable biofuels, such as cellulosic ethanol. The approach used was via surface display of versatile cellulolytic enzyme complexes, namely cellulosomes, on the historical ethanol producer Saccharomyces cerevisiae for simultaneous and synergistic saccharification and fermentation of cellulose to ethanol. The feasibility of assembling cellulosome structures on yeast cell surfaces was first demonstrated by incubating the miniscaffoldin displayed yeasts with the Escherichia coli cell lysates containing three cellulolytic enzymes that were necessary for hydrolyzing cellulose into glucose. The functionally-assembled minicelluosomes retained the synergism for cellulose hydrolysis, resulting in a higher ethanol production level when compared to that obtained from a free cellulase system.;To create a microorganism suitable for a more cost-effective process, called consolidated bioprocessing (CBP), a synthetic consortium capable of displaying mini-cellulosomes on the cell surface via intercellular complementation was subsequently created. In this case, the minicellulosomes were assembled in vivo on yeast surfaces for direct ethanol production and cell growth from cellulose. To tackle the relatively modest ethanol production of the yeast consortium, a designer cellulosme based on the unique feature of the anchoring -adaptor scaffoldin strategy to amplify the number of enzymatic subunits was created. The increased rate in ethanol production indicated that enzyme proximity was crucial to cellulosomal synergy.;To further extend the metabolic engineering strategy toward environmental sustainability, engineered S. cerevisiae strains expressing cysteine desulfhydrase and/or AtPCS were created for enhanced accumulation of arsenic as an efficient biosorbent for environment cleanup.