S91: The RBS Library Calculator: design of synthetic DNA for efficient combinatorial optimization of metabolic pathways

Synthetic metabolic pathways enable microbes to manufacture high-value chemicals from low-value feedstock. However, identifying the optimal enzyme expression levels that maximize a pathway's productivity remains a major challenge. We have developed the RBS Library Calculator, a predictive design method that enables efficient combinatorial optimization of synthetic metabolic pathways. The method combines our thermodynamic model of bacterial translation (Salis et. al., Nature Biotech, 2009) with a genetic optimization algorithm, to computationally determine a RBS library sequence that uniformly varies a protein's expression level across a 100,000-fold scale while minimizing the total library size.

We experimentally demonstrate the RBS Library Calculator's utility on a 5-enzyme biodetoxification pathway that catabolizes furfural, a key inhibitor found in lignocellulosic feedstock. We construct a library of pathway variants that uniformly sample the 5-dimensional enzyme expression level space and then employ selection techniques to identify the pathway variant with the optimal enzyme expression levels. Using this approach, we created a biodetoxification pathway that enables a non-tolerant E. coli strain to succesfully grow on M9 media with 3 g/L furfural (an otherwise toxic condition).