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

封面

如何引用文章

全文:

开放存取 开放存取
受限制的访问 ##reader.subscriptionAccessGranted##
受限制的访问 订阅存取

详细

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.

全文:

受限制的访问

作者简介

Е. Grechishnikova

NRC “Kurchatov Institute”

编辑信件的主要联系方式.
Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

俄罗斯联邦, Moscow

А. Shemyakina

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

俄罗斯联邦, Moscow

А. Novikov

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

俄罗斯联邦, Moscow

Т. Kalinina

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

俄罗斯联邦, Moscow

К. Lavrov

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

俄罗斯联邦, Moscow

А. Yanenko

NRC “Kurchatov Institute”

Email: sel-sanguine@yandex.ru

Kurchatov Genomic Center

俄罗斯联邦, Moscow

参考

  1. Busenlehner L. S., Pennella M. A., Giedroc D. P. The SmtB/ArsR family of metalloregulatory transcriptional repressors: structural insights into prokaryotic metal resistance // FEMS Microbiol. Rev. 2003. V. 27. P. 131‒143.
  2. Coppens L., Wicke L., Lavigne R. SAPPHIRE. CNN: Implementation of dRNA-seq-driven, species-specific promoter prediction using convolutional neural networks // Comput. Struct. Biotechnol. J. 2022. V. 20. P. 4969‒4974.
  3. Dandanell G., Valentin-Hansen P., Erik Løve Larsen J., Hammer K. Long-range cooperativity between gene regulatory sequences in a prokaryote // Nature. 1987. V. 325. P. 823‒826.
  4. Grechishnikova E. G., Shemyakina A. O., Novikov A. D., Lavrov K. V., Yanenko A. S. Rhodococcus: sequences of genetic parts, analysis of their functionality, and development prospects as a molecular biology platform // Crit. Rev. Biotechnol. 2023. V. 43. P. 835‒850.
  5. Jones P., Binns D., Chang H.-Y., Fraser M., Li W., McAnulla C., McWilliam H., Maslen J., Mitchell A., Nuka G. InterProScan 5: genome-scale protein function classification // Bioinformatics. 2014. V. 30. P. 1236‒1240.
  6. Komeda H., Kobayashi M., Shimizu S. Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1 // Proc. Natl. Acad. Sci. USA. 1996. V. 93. P. 4267‒4272.
  7. Lavrov K., Zalunin I., Kotlova E., Yanenko A. A new acylamidase from Rhodococcus erythropolis TA37 can hydrolyze N-substituted amides // Biochemistry (Moscow). 2010. V. 75. P. 1006‒1013.
  8. Lavrov K., Larikova G., Yanenko A. Novel biocatalytic process of N-substituted acrylamide synthesis // Appl. Biochem. Microbiol. 2013. V. 49. P. 702‒705.
  9. Lavrov K., Yanenko A. Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli // Russ. J. Genet. 2013. V. 49. P. 1078‒1081.
  10. Lavrov K., Novikov A., Ryabchenko L., Yanenko A. Expression of acylamidase gene in Rhodococcus erythropolis strains // Russ. J. Genet. 2014. V. 50. P. 1003‒1007.
  11. Lavrov K., Grechishnikova E., Shemyakina A., Novikov A., Kalinina T., Epremyan A., Glinskii S., Minasyan R., Voronin S., Yanenko A. Optimization of the expression of nitrilase from Alcaligenes denitrificans in Rhodococcus rhodochrous to improve the efficiency of biocatalytic synthesis of ammonium acrylate // Appl. Biochem. Microbiol. 2019. V. 55. P. 861‒869.
  12. Lavrov K. V., Shemyakina A. O., Grechishnikova E. G., Novikov A. D., Derbikov D. D., Kalinina T. I., Yanenko A. S. New cblA gene participates in regulation of cobalt-dependent transcription of nitrile hydratase genes in Rhodococcus rhodochrous // Res. Microbiol. 2018. V. 169. P. 227‒236.
  13. Markham N. R., Zuker M. UNAFold: software for nucleic acid folding and hybridization // Methods Mol. Biol. 2008. V. 453. P. 3‒31.
  14. Mukherjee S., Sengupta S. Riboswitch Scanner: an efficient pHMM-based web-server to detect riboswitches in genomic sequences // Bioinformatics. 2016. V. 32. P. 776‒778.
  15. Nawrocki E. P., Eddy S. R. Infernal 1.1: 100-fold faster RNA homology searches // Bioinformatics. 2013. V. 29. P. 2933‒2935.
  16. Novikov A. D., Lavrov K. V., Kasianov A. S., Topchiy M. A., Gerasimova T. V., Yanenko A. S. Complete genome sequence of Rhodococcus sp. strain M8, a platform strain for acrylic monomer production // Microbiol. Resour. Announce. 2021. V. 10. № 10. Art. e01314-20. https://doi.org/10.1128/mra. 01314-20
  17. Nudler E., Mironov A. S. The riboswitch control of bacterial metabolism // Trends Biochem. Sci. 2004. V. 29. P. 11‒17.
  18. Pogorelova T. E., Ryabchenko L. E., Sunzov N. I., Yanenko A. S. Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8 // FEMS Microbiol. Lett. 1996. V. 144. P. 191‒195.
  19. Reese M. G. Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome // Comput. Chem. 2001. V. 26. P. 51‒56.
  20. Reuter J. S., Mathews D. H. RNAstructure: software for RNA secondary structure prediction and analysis // BMC Bioinf. 2010. V. 11. P. 1‒9.
  21. Riabchenko L., Podcherniaev D., Kotlova E., Ianenko A. Cloning the amidase gene from Rhodococcus rhodochrous M18 and its expression in Escherichia coli // Genetika. 2006. V. 42. P. 1075‒1082.
  22. Rice P., Longden I., Bleasby A. EMBOSS: the European molecular biology open software suite // Trends Genet. 2000. V. 16. P. 276‒277.
  23. Shemyakina A. O., Grechishnikova E. G., Novikov A. D., Asachenko A. F., Kalinina T. I., Lavrov K. V., Yanenko A. S. A set of active promoters with different activity profiles for superexpressing Rhodococcus Strain // ACS Synth. Biol. 2021. V. 10. P. 515‒530.
  24. Tatusova T., DiCuccio M., Badretdin A., Chetvernin V., Nawrocki E. P., Zaslavsky L., Lomsadze A., Pruitt K. D., Borodovsky M., Ostell J. NCBI prokaryotic genome annotation pipeline // Nucleic Acids Res. 2016. V. 44. P. 6614‒6624.

补充文件

附件文件
动作
1. JATS XML
下载
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.

下载 (134KB)
索引源数据
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.

下载 (67KB)
索引源数据
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).

下载 (127KB)
索引源数据

版权所有 © Russian Academy of Sciences, 2024