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In vivo Characterization of the Inducible Promoter System of 3-hydroxypropionic Dehydrogenase in Pseudomonas denitrificans

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Abstract

Here, we characterized a gene expression system induced by 3-hydroxypropionic acid (3-HP) in Pseudomonas denitrificans. The system consists of a putative LysR-type transcriptional regulator (LTTR) encoded by hpdR and a LTTR-responsive promoter that positively controls the expression of hpdH, encoding 3-HP dehydrogenase in the presence of 3-HP. In the hpdH-responsive promoter region, two operators exhibiting dyad symmetry and designated O1 and O2 and centered at the −73 and −30 positions, respectively, were located upstream of the hpdH transcription start site. When either O1, O2, or both regions were mutated, the inducibility by the HpdR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcriptional activation. The HpdR protein and its operator sites on hpdH were highly specific for each other, and did not engage in cross-talk with another similar 3-HP-inducible system that is also present in P. denitrificans. This 3-HP-inducible promoter system should be useful in developing biosensors for 3-HP, and/or dynamic gene expression systems for metabolic engineering purposes.

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References

  1. Ongley, S. E., X. Bian, B. A. Neilan, and R. Müller (2013) Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat. Prod. Rep. 30: 1121–1138.

    Article  CAS  Google Scholar 

  2. Kumar, V., S. Ashok, and S. Park (2013) Recent advances in biological production of 3-hydroxypropionic acid. Biotechnol. Adv. 31: 945–961.

    Article  CAS  Google Scholar 

  3. Ashok, S., S. M. Raj, Y. Ko, M. Sankaranarayanan, S. Zhou, V. Kumar, and S. Park (2013) Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT. Metab. Eng. 15: 10–24.

    Article  CAS  Google Scholar 

  4. Ashok, S., M. Sankaranarayanan, Y. Ko, K. E. Jae, S. K. Ainala, V. Kumar, and S. Park (2013) Production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally. Biotechnol. Bioeng. 110: 511–524.

    Article  CAS  Google Scholar 

  5. Zhou, S., S. Ashok, Y. Ko, D. M. Kim, and S. Park (2014) Development of a deletion mutant of Pseudomonas denitrificans that does not degrade 3-hydroxypropionic acid. Appl. Microbiol. Biotechnol. 98: 4389–4398.

    Article  CAS  Google Scholar 

  6. Zhou, S., C. Catherine, C. Rathnasingh, A. Somasundar, and S. Park (2013) Production of 3-hydroxypropionic acid from glycerol by recombinant Pseudomonas denitrificans. Biotechnol. Bioeng. 110: 3177–3187.

    Article  CAS  Google Scholar 

  7. Lim, H. G., M. H. Noh, J. H. Jeong, S. Park, and G. Y. Jung (2016) Optimum rebalancing of the 3-hydroxypropionic acid production pathway from glycerol in Escherichia coli. ACS Synth. Biol. 5: 1247–1255.

    Article  CAS  Google Scholar 

  8. Kwak, S., Y. C. Park, and J. H. Seo (2013) Biosynthesis of 3-hydroxypropionic acid from glycerol in recombinant Escherichia coli expressing Lactobacillus brevis dhaB and dhaR gene clusters and E. coli K-12 aldH. Bioresour. Technol. 135: 432–439.

    Article  CAS  Google Scholar 

  9. Kim, K., S. K. Kim, Y. C. Park, and J. H. Seo (2014) Enhanced production of 3-hydroxypropionic acid from glycerol by modulation of glycerol metabolism in recombinant Escherichia coli. Bioresour. Technol. 156: 170–175.

    Article  CAS  Google Scholar 

  10. Ko, Y., S. Ashok, E. Seol, S. K. Ainala, and S. Park (2015) Deletion of putative oxidoreductases from Klebsiella pneumoniae J2B could reduce 1,3-propanediol during the production of 3-hydroxypropionic acid from glycerol. Biotechnol. Bioprocess Eng. 20: 834–843.

    Article  CAS  Google Scholar 

  11. Kumar, V., M. Sankaranarayanan, M. Durgapal, S. Zhou, Y. Ko, S. Ashok, R. Sarkar, and S. Park (2013) Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol using resting cells of the lactate dehydrogenase-deficient recombinant Klebsiella pneumoniae overexpressing an aldehyde dehydrogenase. Bioresour. Technol. 135: 555–563.

    Article  CAS  Google Scholar 

  12. Raj, S. M., C. Rathnasingh, J. E. Jo, and S. Park (2008) Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain. Process Biochem. 43: 1440–1446.

    Article  CAS  Google Scholar 

  13. Raj, S. M, C. Rathnasingh, W. C. Jung, E. Selvakumar, and S. Park (2010) A Novel NAD+-dependent aldehyde dehydrogenase encoded by the puuC gene of Klebsiella pneumoniae DSM 2026 that utilizes 3-hydroxypropionaldehyde as a substrate. Biotechnol. Bioprocess Eng. 15: 131–138.

    Article  CAS  Google Scholar 

  14. Rathnasingh, C., S. M. Raj, Y. Lee, C. Catherine, S. Ashok, and S. Park (2012) Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains. J. Biotechnol. 157: 633–640.

    Article  CAS  Google Scholar 

  15. Sankaranarayanan, M., S. Ashok, and S. Park (2014) Production of 3-hydroxypropionic acid from glycerol by acid tolerant Escherichia coli. J. Ind. Microbiol. Biotechnol. 41: 1039–1050.

    Article  CAS  Google Scholar 

  16. Zhou, S., S. K. Ainala, E. Seol, T. T. Nguyen, and S. Park (2015) Inducible gene expression system by 3-hydroxypropionic acid. Biotechnol. Biofuels. 8: 169.

    Article  Google Scholar 

  17. Zhou, S., S. M. Raj, S. Ashok, S. Edwardraja, S. G. Lee, and S. Park (2013) Cloning, expression and characterization of 3-hydroxyisobutyrate dehydrogenase from Pseudomonas denitrificans ATCC 13867. PLoS One. 8: e62666.

    Article  CAS  Google Scholar 

  18. Hoffmann, J. and J. Altenbuchner (2015) Functional characterization of the mannitol promoter of Pseudomonas fluorescens DSM 50106 and its application for a mannitol-inducible expression system for Pseudomonas putida KT2440. PLoS One. 10: e0133248.

    Article  Google Scholar 

  19. Maddocks, S. E. and P. C. F. Oyston (2008) Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology (Reading). 154: 3609–3623.

    Article  CAS  Google Scholar 

  20. Schell, M. A. (1993) Molecular biology of the LysR family of transcriptional regulators. Annu. Rev. Microbiol. 47: 597–626.

    Article  CAS  Google Scholar 

  21. Rhee, K. Y., M. Opel, E. Ito, S. Hung, S. M. Arfin, and G. W. Hatfield (1999) Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli. Proc. Natl. Acad. Sci. USA. 96: 14294–14299.

    Article  CAS  Google Scholar 

  22. Lochowska, A., R. Iwanicka-Nowicka, D. Plochocka, and M. M. Hryniewicz (2001) Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control. J. Biol. Chem. 276: 2098–2107.

    Article  CAS  Google Scholar 

  23. Hanko, E. K. R., N. P. Minton, and N. Malys (2017) Characterisation of a 3-hydroxypropionic acid-inducible system from Pseudomonas putida for orthogonal gene expression control in Escherichia coli and Cupriavidus necator. Sci. Rep. 7: 1724.

    Article  Google Scholar 

  24. Rhee, K. Y., D. F. Senear, and G. W. Hatfield (1998) Activation of gene expression by a ligand-induced conformational change of a protein-DNA complex. J. Biol. Chem. 273: 11257–11266.

    Article  CAS  Google Scholar 

  25. Dillon, S. C., E. Espinosa, K. Hokamp, D. W. Ussery, J. Casadesus, and C. J. Dorman (2012) LeuO is a global regulator of gene expression in Salmonella enterica serovar Typhimurium. Mol. Microbiol. 85: 1072–1089.

    Article  CAS  Google Scholar 

  26. Samarasinghe, S., M. S. El-Robh, D. C. Grainger, W. Zhang, P. Soultanas, and S. J. W. Busby (2008) Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR. Nucleic Acids Res. 36: 2667–2676.

    Article  CAS  Google Scholar 

  27. Nguyen, N. H., J. R. Kim, and S. Park (2019) Development of biosensor for 3-hydroxypropionic acid. Biotechnol. Bioprocess Eng. 24: 109–118.

    Article  CAS  Google Scholar 

  28. Rogers, J. K. and G. M. Church (2016) Genetically encoded sensors enable real-time observation of metabolite production. Proc. Natl. Acad. Sci. USA. 113: 2388–2393.

    Article  CAS  Google Scholar 

  29. Rogers, J. K., N. D. Taylor, and G. M. Church (2016) Biosensor-based engineering of biosynthetic pathways. Curr. Opin. Biotechnol. 42: 84–91.

    Article  CAS  Google Scholar 

  30. Ashok, S., S. M. Raj, C. Rathnasingh, and S. Park (2011) Development of recombinant Klebsiella pneumoniae ΔdhaT strain for the co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol. Appl. Microbiol. Biotechnol. 90: 1253–1265.

    Article  CAS  Google Scholar 

  31. Winsor, G. L., E. J. Griffiths, R. Lo, B. K. Dhillon, J. A. Shay, and F. S. L. Brinkman (2016) Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 44: D646–D653.

    Article  CAS  Google Scholar 

  32. Nguyen, N. H., S. K. Ainala, S. Zhou, and S. Park (2019) A novel 3-hydroxypropionic acid-inducible promoter regulated by the LysR-type transcriptional activator protein MmsR of Pseudomonas denitrificans. Sci. Rep. 9: 5333.

    Article  Google Scholar 

  33. Studer, G., C. Rempfer, A. M. Waterhouse, R. Gumienny, J. Haas, and T. Schwede (2020) QMEANDisCo — distance constraints applied on model quality estimation. Bioinformatics. 36: 1765–1771.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This study was financially supported by a grant from the C1 Gas Refinery Program (NRF-2017M3D3A1A01036927-2) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. Trinh Thi Nguyen was financially supported by the BK21 Plus Program of Pusan National University.

The authors declare no conflict of interest.

Neither ethical approval nor informed consent was required for this study.

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Authors and Affiliations

  1. School of Energy and Chemical Engineering, UNIST, Ulsan, 44919, Korea

    Trinh Thi Nguyen, Yeonhee Kim & Sunghoon Park

  2. School of Chemical and Biomolecular Engineering, Pusan National University, Busan, 46241, Korea

    Trinh Thi Nguyen, Jung Rae Kim & Sunghoon Park

  3. NOROO Bio R&D Center, NOROO Holdings Co., Ltd, Suwon, 16229, Korea

    Nam Hoai Nguyen

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  1. Trinh Thi Nguyen
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Nguyen, T.T., Nguyen, N.H., Kim, Y. et al. In vivo Characterization of the Inducible Promoter System of 3-hydroxypropionic Dehydrogenase in Pseudomonas denitrificans. Biotechnol Bioproc E 26, 612–620 (2021). https://doi.org/10.1007/s12257-020-0291-3

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