An Integrative Toolbox for Synthetic Biology in .

The development of microbial cell factories requires robust synthetic biology tools to reduce design uncertainty and accelerate the design-build-test-learn process. Herein, we developed a suite of integrative genetic tools to facilitate the engineering of , a genus of bacteria with considerable biocatalytic potential. We first created pRIME, a modular, copy-controlled integrative-vector, to provide a robust platform for strain engineering and characterizing genetic parts. This vector was then employed to benchmark a series of strong promoters. We found P to be the strongest constitutive rhodococcal promoter, 2.5- to 3-fold stronger than the next in our study, while overall promoter activities ranged 23-fold between the weakest and strongest promoters during exponential growth. Next, we used an optimized variant of P to develop hybrid-promoters and integrative vectors to allow for tetracycline-inducible gene expression in . The best of the resulting hybrid-promoters maintained a maximal activity of ∼50% of P and displayed an induction factor of ∼40-fold. Finally, we developed and implemented a uLoop-derived Golden Gate assembly strategy for high-throughput DNA assembly in . To demonstrate the utility of our approaches, pRIME was used to engineer RHA1 to grow on vanillin at concentrations 10-fold higher than what the wild-type strain tolerated. Overall, this study provides a suite of tools that will accelerate the engineering of for various biocatalytic applications, including the sustainable production of chemicals from lignin-derived aromatics.