ERI: Development of Non-native Sigma Factors for Metabolic Engineering

Miami University, Oxford OH

Investigators

Abstract

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2). Renewable resources are a key element of a sustainable economy. Microbes will play an important role if properly engineered. Product yield increases are needed. Routing material efficiently through different metabolic pathways is critical for increasing yield. The objective of this project is to balance material and energy flows through cell metabolism and product synthesis pathways. A library of sigma factors, which control pathway flows, will be created, and tested. The research will be tied to outreach and workforce development activities. To learn synthetic biology techniques, first year undergraduates and high school teachers will characterize a sigma factor of their choice. Transcriptional balancing of enzyme expression and resource reallocation towards bioproduction are two general strategies that have resulted in enhanced bioproduction. Alternative sigma factors naturally carry out these functions by redirecting transcriptional resources towards new genetic programs in times of stress. Through this work, we will evaluate whether alternative sigma factors will confer bioproduction benefits in E. coli. Mutations to promoter spacer region as well as changes in spacer length will provide transcriptional diversity for a set of non-native sigma factors. A screening approach followed by deep sequencing will determine the identity of promoter variants that give rise to specific transcriptional amounts, improving the prediction of how changes in promoters influence transcription. Libraries of promoters will be applied to heterologous pathways that have multiple side reactions to provide a balanced flux throughout the pathway. Additionally, using a multi-sigma factor-based approach, we will coordinate enzyme expression for complicated pathways that require intermediate build-up to improve specificity. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.