Metabolic pathways are often optimized for pathway flux by reconfiguring promoter strengths driving gene expression as well increasing specific activity of enzymes using protein engineering. In my talk I will show my group's development of a new method to optimize individual enzymes using a pioneering deep sequencing approach that allows interrogation of thousands of enzyme variants in a single pot. Next, I will show application of this method to simultaneously optimize promoter strength and enzyme activity for the conversion of water-soluble pyrolyzed biomass into a range of high-value products. This is important because woody biomass can be threshed and pyrolyzed to create a bio oil. When pyrolyzed in absence of alkali cations and fractionated by water, two streams result: a water-insoluble fraction composed mainly of deconstructed lignin that can be used, after upgrading, as diesel fuel, and a water-soluble fraction chiefly composed of anhydrosugars and small molecular weight lignin. The water-soluble fraction contributes to the instability of the bio oil over time, can degrade engine performance and, hence, should be separated and converted to products that satisfy a different market niche than diesel. Conversion of this water-soluble bio-oil can contribute to the economical processing of woody biomass.

Optimizing pathway flux by simultaneous protein and promoter engineering

247th National Meeting (2014)

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