Structure of the regulatory region of nitrile hydratase genes in Rhodococcus rhodochrous М8, a biocatalyst for production of acrylic heteropolymers

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Abstract

Rhodococcus rhodochrous strain М8 is a platform for development of the biotechnologies for biocatalytic production of acrylic monomers, the raw material for synthesis of acrylic heteropolymers. A genetic system for investigation of the cobalt-dependent transcription of nitrile hydratase genes in this strain was constructed, based the reporter gene of the metal-independent acylamidase from Rhodococcus qingshengii TA37. The cobalt-regulated promoter was shown to be located at a significant distance (~0.5 kb) from nitrile hydratase genes. Excision of the region between the promoter and the nitrile hydratase genes decreased significantly both the promoter activity and the degree of regulation by cobalt. Our results improve the possibilities for rational design of regulated expression cassettes using the promoter of nitrile hydratase genes in Rhodococcus, and for further development of biocatalysts based on these bacteria.

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About the authors

Е. G. Grechishnikova

NRC “Kurchatov Institute”

Author for correspondence.
Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

Russian Federation, Moscow

А. О. Shemyakina

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

Russian Federation, Moscow

А. D. Novikov

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

Russian Federation, Moscow

Т. I. Kalinina

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

Russian Federation, Moscow

К. V. Lavrov

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

Russian Federation, Moscow

А. S. Yanenko

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Structures of DNA loci described in the work: a ‒ genes of the NH cluster in R. rhodochrous M8. nhmC, nhmD are genes of amide-dependent transcriptional regulators of the NH genes, ΔnhmE is a putative open reading frame with unknown function, Pnh569 is the regulatory region of the NH genes, nhmB, nhmA are genes of β and α subunits of NH, respectively, nhmG is the gene of the NH metallochaperone protein, cblA is the gene of the cobalt-dependent transcriptional regulator of the NH genes; b ‒ putative promoters (P1 and P2) and binding sites of the cobalt-dependent regulator cblA (pairs of arrows 1–6) in the regulatory region of Pnh569 upstream of the nhmB gene; c - structure of the NH gene cluster in R. rhodochrous M33 and R. rhodochrous M33 aam strains (in the latter, the nhmBA genes are replaced by the acylamidase gene aam). ΔnhmD - partially deleted nhmD.

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3. Fig. 2. Comparison of the activities of the NG promoter by the levels of transcription and activity of the aam reporter gene: a ‒ acylamidase activities of cells in the R. rhodochrous M33 aam strain grown with and without the addition of CoCl2; b ‒ transcription levels of the Pnh569-nhmBAG operon in R. rhodochrous M8, M33 strains and the Pnh569-aam-nhmG operon in the R. rhodochrous M33 aam strain grown in the presence and absence of CoCl2, determined by RT-qPCR. All data were obtained by selecting cells in the exponential growth phase.

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4. Fig. 3. Structures of deletion variants of the Pnh569 regulatory region (left), acylamidase activities (middle) and relative amounts of aam-nhmG mRNA (right) in R. rhodochrous strains containing the aam reporter gene under the control of these variants. a ‒ Regulatory region variants tested as part of the PnhN-aam-nhmG-cblA expression cassette on the autonomous pRY16 plasmid in the R. rhodochrous M50 strain (stationary cultures); b ‒ regulatory region variants tested as part of the same expression cassette introduced into the chromosome of the R. rhodochrous M33 aam strain (exponentially growing cultures).

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