Metabolic Engineering of Complex Natural Product Pathways in Bacteria

As the “low hanging fruit” of metabolic engineering, primary metabolites were chosen as the initial targets for commercial production in microbial systems. Researchers have since begun to leverage the lessons learned during development of these metabolic engineering approaches with the aim of producing specialized metabolites, which are more complex and challenging targets. Specialized (secondary) metabolites, which are products of complex biochemistries found in nature, include a vast number of chemical candidates (>200,000) for a myriad of applications such as drugs, food additives, consumer products and industrial chemicals. Most of these natural products are chemically complex and heavily functionalized molecules with multiple chiral centers. Nature builds these intricate molecules through multistep biosynthesis using complex promiscuous enzymes such as cytrochrome P450’s. In addition to the low accumulation of these molecules in nature (at ppm levels), the structural complexity of natural products precludes the development of economical synthetic routes to these molecules. Therefore, developing tools and technologies for the rapid and efficient construction of multi-step biosynthetic pathways enabling the creation of microbial strains capable of producing specialized natural products is a high priority research area for the metabolic engineering community.

We developed a new metabolic engineering approach, multivariate modular metabolic engineering (MMME), for systematically engineering such complex pathways. Recently, we expanded the scope of our approach to enable quick construction and optimization of orthologous pathways for the selection of the best variant biosynthetic routes to desired specialized products. These approaches enabled not only rapid construction of microbial strains for synthesizing complex biochemicals in useful accessible quantities, but provided several key insights on natural product biosynthesis and the origins of biosynthetic diversity of these specialized products. Here, we focus on the application of pathway engineering, targeted proteomics, metabolomics and transcriptomics in the context of MMME for optimizing the multi-step pathways for terpenoid synthesis in bacteria. This enabled the rapid construction of hundreds of strains, with lines capable of reaching multigram per liter production of several key natural product chemicals.