Development of Saccharomyces Cerevisiae Strains for Continuous Bioprocessing at Low pH

The successful implementation of a biorefinery for chemical processing could lead to the eventual replacement of petroleum-based feedstocks with renewable resources. Because this concept requires the coordinated integration of many fermentation units, it would be advantageous to use continuous operations. Consequently, it is necessary to construct process organisms that will not undergo significant genetic modification throughout the continuous process. Furthermore, because many common biological products are organic acids, fermentation at low pH would be desirable. This project will determine the technical feasibility of generating enhanced biocatalysts specifically designed for continuous bioprocessing at low pH. The approach involves the development of strains of Saccharomyces cerevisiae, using the 1,4-diacids (succinic, fumaric, and malic) as exemplary products. The OptKnock computational framework (developed to suggest gene deletion strategies that couple biochemical production to cell growth, leading to genetically-stable overproducing microorganisms) will be applied to enumerate sets of multiple gene deletions that lead to growth-coupled production of the 1,4-diacids. The four most promising strategies will be constructed, and the resulting strains will be subjected to adaptive evolution, in order to enhance their rates of growth and production, and to improve their tolerance to low pH. Finally, the strains will be characterized in a continuous culture. The overall measures of success will be the productivity and yield of the generated strains, the genetic stability of cultures in a long-term chemostat, and the ability to operate at pH below 4.0. Commercial Applications and other Benefits as described by the awardee: The successful completion of this project should result in a set of microbial production strains for several commodity chemicals. These strains not only should have yield and productivity potential that exceeds current fermentation technology, but also should be genetically stable and thus suitable for continuous processing. This would represent a significant step toward the development of an economically-viable biorefinery.