(5bo) Synergy of Protein and Genome Engineering

The focus of my work has been to use protein and metabolic engineering to produce industrially important chemicals and fuels from biomass. At the same time, I have developed genome engineering tools to facilitate studies in metabolic engineering, synthetic biology, and systems biology.

1) Selective catalysis for xylitol production: We have engineered a highly substrate specific D-xylose reductase (XR) that retains very little of its natural activity toward the epimeric substrate L-arabinose. Using structure-function analysis and directed evolution, we evolved this mutant with >16-fold preference for xylose compared to arabinose. Whole cell biocatalysis studies demonstrate that our mutant produces very little byproduct (arabinitol) from a mixture of hemicellulosic sugars, while producing xylitol with near-wild-type productivity.

2) A tool for E. coli genome modification: As one of the most important platform organism for biocatalysis, there is always room for better methods for E. coli genome manipulation. However, the eccentricities of its recombination machinery have necessitated the use of heterologous (phage) proteins for quick, effective genome modification. We have developed a tool for easily modifying the E. coli genome that does not rely on any heterologous proteins. PCR products are directly used as mutagenic cassettes and recombinants with desired deletion or point mutation can be obtained with high frequency.

3) Biobutanol from S. cerevisiae: Butanol has been identified as an alternative to ethanol due to several superior properties as a liquid biofuel. Heterologous production in yeast was challenging until we performed metabolic engineering on the expression strain and directed evolution on one of the butanol biosynthetic enzymes for soluble expression. The synergy of the two efforts finally yielded a strain capable of producing butanol. Our efforts demonstrate that yeast is a viable platform organism to produce butanol from glucose.

4) A tool for S. cerevisiae genome modification: S. cerevisiae is a model organism for industrial biocatalysis. We developed a tool for quick, precise modification of the yeast genome. It is a powerful, yet simple, tool for point mutagenesis, insertions, and deletion studies alike. Named MIRAGE (mutagenic inverted repeat assisted genome engineering), this method uses yeast's highly efficient homologous recombination machinery to integrate a mutagenic cassette containing an inverted repeat (IR) into the chromosome. Due to its inherent instability in the chromosome, the IR catalyzes its own excision leaving behind only the desired mutation without any scars at very high frequency.