Assembly of multi-gene pathways and combinatorial pathway libraries through ePathBrick vectors.

As an emerging discipline, synthetic biology is becoming increasingly important to design, construct, and optimize metabolic pathways leading to desired phenotypes such as overproduction of biofuels and pharmaceuticals in genetically tractable organisms. We have recently developed a versatile gene assembly platform ePathBricks supporting the modular assembly of multi-gene pathway components and combinatorial generation of pathway diversities. In this protocol, we will detail the process to assemble a seven gene flavonoid pathway (~9 kb) on one single ePathBrick vector. We will also demonstrate that a three-gene flavonoid pathway can be easily diversified to 54 pathway equivalents differing in pathway configuration and gene order; coupled with high-throughput screening techniques, we envision that this combinatorial strategy would greatly improve our ability to exploit the full potential of microbial cell factories for recombinant metabolite production.