Development of a genetic toolset for the highly engineerable and metabolically versatile Acinetobacter baylyi ADP1

One primary objective of synthetic biology is to improve the sustainability of chemical manufacturing. Biological systems can utilize a variety of carbon sources, including waste streams that pose challenges to traditional chemical processing such as lignin biomass, providing opportunity for remediation and valorization of these materials. Success, however, depends on identifying microorganisms that are both metabolically versatile and engineerable. This has been a historic hindrance. Here, we leverage the facile genetics of the metabolically versatile bacterium Acinetobacter baylyi ADP1 to create easy and rapid molecular cloning workflows, a promoter library, ribosomal binding site (RBS) variants, and an unprecedented number of bacterial chromosomal protein expression sites and variants. Moreover, we demonstrate the utility of these tools by examining ADP1’s catabolic repression regulation, creating a strain with improved potential for lignin bioprocessing. Taken together, this work establishes ADP1 as an ideal host for a variety of sustainability and synthetic biology applications.