Towards Generalized Platforms for Functional Genomics in α-Proteobacteria

Authors:

Ashley N. Hall¹ ([email protected]), Kyle Kinney¹, Amy B. Banta^1,2, Timothy Donohue^1,2, and Jason M. Peters^1,2

Institutions:

¹DOE Great Lakes Bioenergy Research Center; and ²University of Wisconsin–Madison

Goals

Enable rational engineering of alphaproteobacteria that can synthesize biofuels and bioproducts from lignocellulosic biomass. This project develops and validates genomic insertion sites, inducible promoters, and inducible riboswitches for bacteria such as Novosphingobium aromaticivorans, Zymomonas mobilis, and Rhodobacter sphaeroides.

Abstract

Numerous alphaproteobacterial species have promising traits for converting lignocellulosic biomass to useful biofuels and bioproducts. However, many of the genes required to carry out these roles have remained enigmatic in part due to limited genetic tools developed for these organisms. Next-generation genetic tools, such as CRISPRi-seq, are capable of systematically phenotyping all genes, but they have not been broadly deployed in alphaproteobacteria. Building on success in establishing genome-scale CRISPRi in Z. mobilis, the team seeks to develop generalized platforms for synthetic biology and functional genomics in alphaproteobacteria. Here, researchers lay the groundwork for these platforms by optimizing site specific integration and developing synthetic, inducible promoters for N. aromaticivorans and R. sphaeroides. These enhanced genetic tools will enable basic and applied research such as delivery of CRISPRi systems to investigate gene function and expression of heterologous pathways to generate valuable bioproducts.

Funding Information

This material is based upon work supported by the Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under Award Number DE-SC0018409.