The number of small molecules that can be produced by engineered microbes has rapidly increased in recent years. This increase can be attributed to the development of recombinant DNA technology and advances in metabolic engineering and directed evolution. The current trend in engineering microbes utilizes an iterative approach where genetic perturbations are made, metabolite production is evaluated, and new designs are implemented. The next generation is going to take advantage of both systems and synthetic biology to generate increasingly complex perturbations and clearer pictures of cellular physiology.

Within the Great Lakes Bioenergy Research Center (GLBRC), we are utilizing this strategy to develop the next generation of biofuel producers. Our strategy is to develop first-pass panels of biofuel producing strains based on a minimal set of mutations chosen with the assistance of metabolic models. These panels are screened for biofuel production, and the best strains are selected for REDIME (REiterative DIrected Microbial Evolution). The REDIME process entails growing the best candidate strains at larger scales, sampling the cultures for multi-omic analysis, identifying limiting genetic elements, designing/evolving new strains that circumvent the limitations, and evaluating the improvements before repeating the cycle. In order to implement this strategy, the GLBRC has assembled two facilities, a high throughput microbial phenotype screening facility, and a bioenergy chemostat facility. These facilities have the capability to screen panels of strains and generate sufficient biomass for multi-omic analysis. In addition, GLBRC researchers have developed pipelines for sequencing, transcriptomic, proteomic, metabolomic, and other systems-level studies. The goal of these pipelines is to decrease the turnaround between fermentation and analysis, such that the REDIME iteration time is as short as possible.

This talk will use examples from preliminary and published experiments to describe how the elements of synthetic and systems biology can be integrated into the REDIME process for biofuel production. Recent work has used systems biology to evaluate multi-omic data and to identify sources of toxicity during the production of biofuels. Subsequently, synthetic biology techniques afforded the ability to overcome these toxic effects by regulating metabolic circuits involved in the biofuel pathways. The combinations of systems biology and synthetic biology permit increasingly sophisticated levels of control over cellular processes. These methods are more robust than traditional gene overexpression and or deletions. I will also briefly discuss the development of new synthetic biology tools and their use in biofuel development.