Engineering microbial co-cultures for complex natural product biosynthesis

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Many naturally-occurring pharmaceutical molecules (for example: antibiotics, anti-cancer drugs) are the result of a long sequence of reactions that add, piece by piece, different subunits to the growing molecule, in a fashion similar to an industrial assembly line. When production occurs in a single organism, it places a large burden on the organism to provide all of the raw materials and energy needed to complete construction. This project will investigate a strategy that creates organisms capable of efficiently producing a particular subunit of the larger drug molecule, "subcontracts" production of subunits to multiple organisms, then brings the subunits together to create the final product. If successful, this could dramatically improve the efficiency of large molecule drug production, and could also increase the speed of identification of more effective drug molecules. This project will also expand the development of a STEM-trained workforce through targeted outreach and engagement of high school and college students from under-represented populations in the research project.This proposed study aims to adapt engineered E. coli strains to constitute co-cultures for production of three representative natural products with characteristically long and complicated biosynthetic pathways. Each biosynthetic pathway is divided into two independent modules, each of which is accommodated in a different, specialized E. coli strain. Such designed pathway modularization allows for reduction of metabolic burden and improvement of biosynthetic performance of the co-culture strains, as each strain is only responsible for one portion of the biosynthesis. The involved strains will also be metabolically engineered individually to meet the needs of the designated pathway enzymes and maximize their bioconversion capabilities. Moreover, the biosynthetic strength of the individual pathway modules will be balanced for bioproduction optimization through manipulation of the relative ratio of the co-culture populations. The microbial systems will be scaled up to investigate population stability and bioproduction behavior under high cell density conditions. This project offers outstanding potential to design and engineer the production of complex molecules. The fundamental knowledge gained regarding the dynamic regulation of population ratios and metabolic activity in mixed populations will provide insight into the behavior of natural microbial mixed populations, which will have implications for a wide variety of industrial and environmentally-important processes.