S2: Pathway engineering via synthetic biology

Microbial synthesis of chemicals and fuels typically depends upon the creation or introduction of heterologous metabolic pathways into the production hosts.  Balancing the flux of a heterologous metabolic pathway is difficult yet critical to the success of metabolic engineering.  One prominent example is the metabolic engineering of a Saccharomyces cerevisiae strain harboring a heterologous xylose utilizing pathway for cellulosic biofuels production, which remains a challenge even after more than three decades of research.  Here we report the development of synthetic biology approaches for rapid creation of highly efficient xylose utilizing pathways for ethanol production.  Our first approach is to explore various combinations of the enzyme homologues with different properties.  A library of more than eight thousand xylose utilization pathways was generated using DNA assembler followed by multi-tiered screening, which led to the identification of a number of strain-specific combinations of the enzymes for efficient conversion of xylose to ethanol.  Our second approach is to explore various combinations of the promoters with varying strengths for each pathway gene.  Specifically, a library of mutant pathways was created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism using the DNA assembler method followed by high throughput screening/selection.  After a single round of library creation and screening, we were able to generate xylose (or cellobiose) utilizing pathways with near-highest (or highest) efficiency ever reported in both laboratory and industrial yeast strains.  Interestingly, the engineered xylose or cellobiose utilizing pathways using both approaches were all strain-specific.