Construction of Genetic Logic Gates Based on the T7 RNA Polymerase Expression System in Rhodococcus opacus PD630

Rhodococcus opacus PD630 (R. opacus) is a nonmodel, Gram-positive bacterium that holds promise as a biological catalyst for the conversion of lignocellulosic biomass to value-added products. In particular, it demonstrates both a high tolerance for and an ability to consume inhibitory lignin-derived aromatics, generates large quantities of lipids, exhibits a relatively rapid growth rate, and has a growing genetic toolbox for engineering. However, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Furthermore, genetic logic circuits for sophisticated gene regulation have never been demonstrated in Rhodococcus spp. To address these shortcomings, two inducible T7 RNA polymerase-based expression systems were implemented for the first time in R. opacus and applied to the construction of AND and NAND genetic logic gates. Additionally, three isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoters were created by inserting LacI binding sites into newly characterized constitutive promoters. Furthermore, four novel aromatic sensors for 4-hydroxybenzoic acid, vanillic acid, sodium benzoate, and guaiacol were developed, expanding the gene expression toolbox. Finally, the T7 RNA polymerase platform was combined with a synthetic IPTG-inducible promoter to create an IMPLY logic gate. Overall, this work represents the first demonstration of a heterologous RNA polymerase system and synthetic genetic logic in R. opacus, enabling complex and tunable gene regulation in this promising nonmodel host for bioproduction.