Tools for Genetic Engineering and Gene Expression Control in and .

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, , , and , show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for , and still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for and to address these issues. We demonstrate that Tn transposition is an effective approach for introducing engineered DNA into the chromosome of and . We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn integration with promoters from our library, we establish CRISPR interference systems for and that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria.