A modular toolkit for environmentalRhodococcus, Gordonia, andNocardiaenables complex metabolic manipulation

Abstract

ABSTRACTSoil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome, but largely lack genetic tools comparable to those available in model species such asE. coliorPseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolateRhodococcus ruberC208 and demonstrate conserved functionality in diverse environmental isolates ofRhodococcus, NocardiaandGordonia. This toolkit encompasses Mycobacteriale origins of replication, broad-host range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway inRhodococcus erythropolisN9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux towards the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species.IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens.Rhodococcus, GordoniaandNocardiaare three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these,Rhodococcus ruberC208 was shown to degrade polyethylene,Gordonia paraffinivoranscan assimilate carbon from solid hydrocarbons, andNocardia neocaledoniensis(and many otherNocardia) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.