Advances in DNA reading and writing technologies are driving adoption of new paradigms for engineering of biology. Designing and engineering microbes that produce valuable chemicals and fuels in a cost-effective and sustainable fashion is no longer limited by our ability to rewire living cells. The current challenge is to understand the complex microbial genetic machinery to be able to decide which genetic parts must be rewired to achieve the desired bioproducts. Genetic changes that affect gene regulatory networks are known to have the largest effects in biological function. Therefore, this project will develop a new standard for the engineering of microbial systems that is based on the rational design, engineering, and optimization of regulatory networks. Computer-aided design platforms will guide the assembly of synthetic genetic networks. These design platforms will be first applied to model organisms to then transfer the technologies to photosynthetic and anaerobic bacteria, as well as stress-tolerant yeast. Ultimately, this research will enable predictable and dynamic control of multiple economically important traits such growth on plant-derived feedstocks, tolerance to toxic byproducts and other stresses, and production of multiple and complex target molecules. By deploying these new biological engineering platforms for non-model microorganisms with potential industrial relevance, this project will advance toward DOE's mission in the development of sustainable biofuel production from renewable sources.