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

该奖项全部或部分由2021年美国救援计划法案（公法117-2）资助。可再生资源是可持续经济的关键要素。 微生物将发挥重要作用，如果适当的工程。需要提高产品产量。通过不同的代谢途径有效地路由材料是提高产量的关键。该项目的目标是通过细胞代谢和产物合成途径平衡物质和能量流。 将创建并测试控制路径流的sigma因子库。 这项研究将与外联和劳动力发展活动挂钩。为了学习合成生物学技术，一年级的本科生和高中教师将描述他们选择的西格玛因子。酶表达的转录平衡和资源向生物生产的重新分配是两个一般的策略，导致提高生物生产。替代的σ因子通过在压力下将转录资源重定向到新的遗传程序来自然地执行这些功能。通过这项工作，我们将评估是否替代西格玛因素将赋予生物生产的好处，在E。杆菌启动子间隔区的突变以及间隔区长度的变化将为一组非天然σ因子提供转录多样性。随后进行深度测序的筛选方法将确定产生特定转录量的启动子变体的身份，从而改善对启动子变化如何影响转录的预测。启动子文库将应用于具有多种副反应的异源途径，以在整个途径中提供平衡的通量。此外，我们还将使用基于多西格玛因子的方法，协调酶在需要中间体构建以提高特异性的复杂途径中的表达。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.