Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630

DeLorenzo, Drew; Moon, Tae Seok

doi:10.1021/acssynbio.9b00213

Construction of genetic logic gates based on the T7 RNA polymerase expression system in Rhodococcus opacus PD630

Journal Article · Tue Jul 30 00:00:00 EDT 2019 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.9b00213· OSTI ID:1545618

DeLorenzo, Drew ^[1]; Moon, Tae Seok ^[2]

Washington Univ., St. Louis, MO (United States); Washington University in St. Louis
Washington Univ., St. Louis, MO (United States)

Rhodococcus opacus PD630 (R. opacus) is a non-model, gram-positive bacterium which holds promise as a biological catalyst for the conversion of lignocellulosic biomass into 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. Yet, the availability of genetic parts for tunable, high-activity gene expression is still limited in R. opacus. Moreover, 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 constructing AND and NAND genetic logic gates. Additionally, three 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. Lastly, 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 non-model host for bioproduction.

View Accepted Manuscript (DOE)

Research Organization:: Washington Univ., St. Louis, MO (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: SC0018324

OSTI ID:: 1545618

Journal Information:: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 8 Vol. 8; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Boolean logic
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References (52)

Cited By (2)

Figures / Tables (6)

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