Synthetic biology and metabolic engineering are promising fields that provides methodologies for microbial production of useful compounds. The target product is not limited to natural chemicals discovered in nature; non-natural chemicals could also be “bio”-synthesized by engineered enzymes that acquired new functions. However, novel biological functions engineered in the laboratory are often weak and unstable, which becomes an obstacle for further engineering and applications. Our group are developing genetic engineering tools and methodologies for microorganisms to accelerate the construction of non-natural biosynthetic pathways in the laboratory.