(450c) A Modular Synthetic Biology Toolkit for Environmental Actinobacteria

Actinobacteria are a broad group of Gram positive, high GC content bacteria, which are widely distributed in soils and aquatic environments. While genera such as Streptomyces are familiar as the source of many antibiotics, and Mycobacterium are well-studied as the causative agents of human and livestock disease, our knowledge of the wider phylum and the genetic tools available to interrogate them remain comparatively limited. However, recent advances in genome sequencing and several taxonomic revisions have shed new light on these species. In particular, the mycolata taxon, which is defined by the presence of mycolic acid as a component of the cell wall, is now thought to possess vast, untapped potential as new hosts for the bioproduction of commodity chemicals, biodegradation of natural and synthetic polymers, and a source of novel secondary metabolites including polyketide antimicrobials. Unfortunately, although this taxon includes Mycobacterium which has a large research community and established genetic tools, few of these have proven directly transferable to the other members of interest, particularly genera such as Rhodococcus, Nocardia, Gordonia, and Dietzia.

To facilitate the use of these bacteria as hosts for metabolic engineering, we have built a new, modular DNA assembly toolkit for constructing plasmids and genetic circuits which maintains broad compatibility across a wide range of environmental mycolata isolates. This toolkit comprises six plasmid origins of replication, five different selectable markers, a gradient series of synthetic promoters and universal, bicistronic ribosome binding sites, and various transcriptional logic. Collectively, these tools enable the construction of robust genetic circuits in multiple genera with previously unattainable control of gene expression. In addition, this toolkit has been designed to maximize the use of common parts between these genera and E. coli, where most plasmid assembly is expected to occur due to the ease of genetic manipulation. This dramatically reduces the size and complexity of the component DNA parts required and is expected to improve genetic stability during cloning. Using this toolkit, we demonstrate genetic circuits for metabolic engineering of high-value isoprenoid biosynthesis, protein engineering to enable novel carbon assimilation pathways, and modular assembly of transposons for genome-wide mutagenesis.