An Integrative Toolbox for Synthetic Biology in Rhodococcus

Abstract

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 Rhodococcus, 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 M6 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 M6 to develop hybrid-promoters and integrative vectors to allow for tetracycline-inducible gene expression in Rhodococcus. The best of the resulting hybrid-promoters maintained a maximal activity of ∼50% of P M6 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 Rhodococcus. To demonstrate the utility of our approaches, pRIME was used to engineer Rhodococcus jostii 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 Rhodococcus for various biocatalytic applications, including the sustainable production of chemicals from lignin-derived aromatics.

Applications of Serine Integrases in Synthetic Biology over the Past Decade

“…As previously reported, serine integrases were active in a wide range of hosts, including not only prokaryotes (E. coli [66], Pseudomonas [104], Rhodococcus [105], and other nonmodel bacteria [56]), but also eukaryotes (Saccharomyces cerevisiae [67] and mammalian cell lines [68][69][70][71]106,107]), animals (Drosophila [63] and mouse [72]), and plants (tobacco [62,73] and Arabidopsis [61,74]) (Figure 8, top). However, it is necessary to provide other carriers for microorganisms to resist extreme environments for practical applications such as capsules for engineering living therapeutics [64], hydrogels for bacteria protection [108], and in situ DNA brushes [60] for memory materials design (Figure 8, bottom left).…”

A myo-inositol dehydrogenase involved in aminocyclitol biosynthesis of hygromycin A

“…We found that Hyg17 formed inclusion bodies when recombinantly produced by various E. coli expression strains. However, we were able to obtain pure soluble protein when expressing Hyg17 in a Rhodococcus expression system ( Supporting Information File 1 , Figure S1) [ 10 – 11 ]. According to the proposed hygromycin A biosynthetic pathway, Hyg17 is a myo -inositol dehydrogenase.…”

“…hyg17 was cloned into pTip-QC1 [ 10 ] using NdeI and XhoI restriction sites and verified by DNA sequencing (Eurofins Genomics). pTip-QC1- hyg17 plasmid [ 10 ] was transformed into Rhodococcus jostii RHA1 [ 11 ]. Cultures were grown in Luria Bertani (LB) media supplemented with 34 µg mL −1 chloramphenicol at 30 °C while shaking at 200 rpm for 48 h reaching an OD 600 of ≈1.4 then induced with 50 µL of 20 mg mL −1 thiostrepton and grown for another 24 h at 30 °C while shaking at 200 rpm.…”

Deciphering the biosynthesis of a novel lipid in Mycobacterium tuberculosis expands the known roles of the nitroreductase superfamily

“…RHA1 is a fast-growing mycolic acid-producing actinobacterium that lacks a homologous TyzABC system. The acyltransferase was expressed using two different systems: a pTip thiostrepton-inducible expression plasmid ( 29 ) and an integrative vector, pRIME, under the control of a strong constitutive promoter ( 30 ). Metabolites were extracted from cell pellets and supernatants of stationary phase cultures using ethyl acetate.…”