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Engineering the Translational Machinery for Biotechnology Applications

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Abstract

The ribosome is an essential organelle in charge of the translational processes in all kinds of cells. Currently, the scenario of its function has been significantly expanded from the classic machine for protein synthesis to a regulatory platform for quality control to maintain the protein homeostasis in a living cell. The ribosome is much more than a mechanical device with a static structure: it is inherently dynamic in structure and function, especially in response to the environmental fluctuations. Considerable effort has been made to regulate its structure and physiological function by engineering the components of a ribosome. The findings of the pioneering studies significantly deepened our understanding of a ribosome and exemplified how a ribosome could be engineered for biotechnology purposes in the era of synthetic biology. The engineering of ribosome offered highly accessible methods capable of comprehensively optimizing the performance of strains of industrial importance. In this article, the relevant recent advances were systematically reviewed.

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References

  1. Bozhuyuk, K. A. J., Fleischhacker, F., Linck, A., Wesche, F., Tietze, A., Niesert, C. P., et al. (2018). De novo design and engineering of non-ribosomal peptide synthetases. Nature Chemistry,10, 275–281.

    PubMed  Google Scholar 

  2. Palade, G. E. (1955). A small particulate component of the cytoplasm. Journal of Biophysical and Biochemical Cytology,1, 59–68.

    CAS  PubMed  Google Scholar 

  3. Capel, M. S., Engelman, D. M., Freeborn, B. R., Kjeldgaard, M., Langer, J. A., Ramakrishnan, V., et al. (1987). A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli. Science,238, 1403–1406.

    CAS  PubMed  Google Scholar 

  4. Ban, N., Nissen, P., Hansen, J., Moore, P. B., & Steitz, T. A. (2000). The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science,289, 905.

    CAS  PubMed  Google Scholar 

  5. Bashan, A., Agmon, I., Zarivach, R., Schluenzen, F., Harms, J., Berisio, R., et al. (2003). Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression. Molecular Cell,11, 91–102.

    CAS  PubMed  Google Scholar 

  6. Wada, A. (1998). Growth phase coupled modulation of Escherichia coli ribosomes. Genes to Cells,3, 203–208.

    CAS  PubMed  Google Scholar 

  7. Wada, A., Mikkola, R., Kurland, C. G., & Ishihama, A. (2000). Growth phase-coupled changes of the ribosome profile in natural isolates and laboratory strains of Escherichia coli. Journal of Bacteriology,182, 2893–2899.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Slavov, N., Semrau, S., Airoldi, E., Budnik, B., & van Oudenaarden, A. (2015). Differential stoichiometry among core ribosomal proteins. Cell Reports,13, 865–873.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kondrashov, N., Pusic, A., Stumpf, C. R., Shimizu, K., Hsieh, A. C., Ishijima, J., et al. (2011). Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell,145, 383–397.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Degenhardt, R. F., & Bonham-Smith, P. C. (2008). Arabidopsis ribosomal proteins RPL23aA and RPL23aB are differentially targeted to the nucleolus and are disparately required for normal development. Plant Physiology,147, 128–142.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Kim, K. Y., Park, S. W., Chung, Y. S., Chung, C. H., Kim, J. I., & Lee, J. H. (2004). Molecular cloning of low-temperature-inducible ribosomal proteins from soybean. Journal of Experimental Botany,55, 1153–1155.

    CAS  PubMed  Google Scholar 

  12. Simsek, D., & Barna, M. (2017). An emerging role for the ribosome as a nexus for post-translational modifications. Current Opinion in Cell Biology,45, 92–101.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Nesterchuk, M. V., Sergiev, P. V., & Dontsova, O. A. (2011). Posttranslational modifications of ribosomal proteins in Escherichia coli. Acta Naturae,3, 22–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Genuth, N. R., & Barna, M. (2018). The discovery of ribosome heterogeneity and its implications for gene regulation and organismal life. Molecular Cell,71, 364–374.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Xue, S., & Barna, M. (2012). Specialized ribosomes: a new frontier in gene regulation and organismal biology. Nature Reviews Molecular Cell Biology,13, 355–369.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., & Mankin, A. S. (2015). Protein synthesis by ribosomes with tethered subunits. Nature,524, 119–124.

    CAS  PubMed  Google Scholar 

  17. Kannan, K., Tsvetanova, B., Chuang, R. Y., Noskov, V. N., Assad-Garcia, N., Ma, L., et al. (2016). One step engineering of the small-subunit ribosomal RNA using CRISPR/Cas9. Science Reports,6, 30714.

    CAS  Google Scholar 

  18. Vesper, O., Amitai, S., Belitsky, M., Byrgazov, K., Kaberdina, A. C., Engelberg-Kulka, H., et al. (2011). Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli. Cell,147, 147–157.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Luidalepp, H., Berger, S., Joss, O., Tenson, T., & Polacek, N. (2016). Ribosome shut-down by 16S rRNA fragmentation in stationary-phase Escherichia coli. Journal of Molecular Biology,428, 2237–2247.

    CAS  PubMed  Google Scholar 

  20. Oron-Gottesman, A., Sauert, M., Moll, I., & Engelberg-Kulka, H. (2016). A Stress-induced bias in the reading of the genetic code in Escherichia coli. MBio,7, e01855.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Nikolic, N., Didara, Z., & Moll, I. (2017). MazF activation promotes translational heterogeneity of the grcA mRNA in Escherichia coli populations. PeerJ,5, e3830.

    PubMed  PubMed Central  Google Scholar 

  22. Nikolic, N., Bergmiller, T., Vandervelde, A., Albanese, T. G., Gelens, L., & Moll, I. (2018). Autoregulation of mazEF expression underlies growth heterogeneity in bacterial populations. Nucleic Acids Research,46, 2918–2931.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Mets, T., Kasvandik, S., Saarma, M., Maivali, U., Tenson, T., & Kaldalu, N. (2019). Fragmentation of Escherichia coli mRNA by MazF and MqsR. Biochimie,156, 79–91.

    CAS  PubMed  Google Scholar 

  24. Kohanski, M. A., Dwyer, D. J., & Collins, J. J. (2010). How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology,8, 423–435.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Funatsu, G., & Wittmann, H. G. (1972). Ribosomal proteins. 33. Location of amino-acid replacements in protein S12 isolated from Escherichia coli mutants resistant to streptomycin. Journal of Molecular Biology,68, 547–550.

    CAS  PubMed  Google Scholar 

  26. Finken, M., Kirschner, P., Meier, A., Wrede, A., & Bottger, E. C. (1993). Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot. Molecular Microbiology,9, 1239–1246.

    CAS  PubMed  Google Scholar 

  27. Honore, N., & Cole, S. T. (1994). Streptomycin resistance in mycobacteria. Antimicrobial Agents and Chemotherapy,38, 238–242.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Meier, A., Kirschner, P., Bange, F. C., Vogel, U., & Bottger, E. C. (1994). Genetic alterations in streptomycin-resistant Mycobacterium tuberculosis: mapping of mutations conferring resistance. Antimicrobial Agents and Chemotherapy,38, 228–233.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Shima, J., Hesketh, A., Okamoto, S., Kawamoto, S., & Ochi, K. (1996). Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2). Journal of Bacteriology,178, 7276–7284.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Suzuki, T., Seta, K., Nishikawa, C., Hara, E., Shigeno, T., & Nakajima-Kambe, T. (2015). Improved ethanol tolerance and ethanol production from glycerol in a streptomycin-resistant Klebsiella variicola mutant obtained by ribosome engineering. Bioresource Technology,176, 156–162.

    CAS  PubMed  Google Scholar 

  31. Chen, L., Shang, G., Yuan, W., Wu, Y., & Bai, F. (2012). Screening of Clostridium strains through ribosome engineering for improved butanol production. Sheng Wu Gong Cheng Xue Bao,28, 1048–1058.

    CAS  PubMed  Google Scholar 

  32. Ochi, K., Okamoto, S., Tozawa, Y., Inaoka, T., Hosaka, T., Xu, J., et al. (2004). Ribosome engineering and secondary metabolite production. Advances in Applied Microbiology,56, 155–184.

    CAS  PubMed  Google Scholar 

  33. Zhu, S., Duan, Y., & Huang, Y. (2019). The application of ribosome engineering to natural product discovery and yield improvement in Streptomyces. Antibiotics (Basel). https://doi.org/10.3390/antibiotics8030133.

    Article  PubMed Central  Google Scholar 

  34. Wang, L., Chen, X., Wu, G., Li, S., Zeng, X., Ren, X., et al. (2017). Enhanced epsilon-poly-L-lysine production by inducing double antibiotic-resistant mutations in Streptomyces albulus. Bioprocess and Biosystems Engineering,40, 271–283.

    CAS  PubMed  Google Scholar 

  35. Tanaka, Y., Kasahara, K., Izawa, M., & Ochi, K. (2017). Applicability of ribosome engineering to vitamin B12 production by Propionibacterium shermanii. Bioscience, Biotechnology, and Biochemistry,81, 1636–1641.

    CAS  PubMed  Google Scholar 

  36. Kurosawa, K., Hosaka, T., Tamehiro, N., Inaoka, T., & Ochi, K. (2006). Improvement of alpha-amylase production by modulation of ribosomal component protein S12 in Bacillus subtilis 168. Applied and Environment Microbiology,72, 71–77.

    CAS  Google Scholar 

  37. Xu, J., Tozawa, Y., Lai, C., Hayashi, H., & Ochi, K. (2002). A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2). Molecular Genetics and Genomics,268, 179–189.

    CAS  PubMed  Google Scholar 

  38. Halfon, Y., Jimenez-Fernandez, A., La Rosa, R., Espinosa Portero, R., Krogh Johansen, H., Matzov, D., et al. (2019). Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate. Proceedings of the National Academy of Sciences of the United States of America,116, 22275–22281.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhao, Y., Song, Z., Ma, Z., Bechthold, A., & Yu, X. (2019). Sequential improvement of rimocidin production in Streptomyces rimosus M527 by introduction of cumulative drug-resistance mutations. Journal of Industrial Microbiology and Biotechnology,46, 697–708.

    CAS  PubMed  Google Scholar 

  40. Tamehiro, N., Hosaka, T., Xu, J., Hu, H., Otake, N., & Ochi, K. (2003). Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus. Applied and Environment Microbiology,69, 6412–6417.

    CAS  Google Scholar 

  41. Okamoto-Hosoya, Y., Hosaka, T., & Ochi, K. (2003). An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2). Microbiology,149, 3299–3309.

    CAS  PubMed  Google Scholar 

  42. Hosaka, T., Xu, J., & Ochi, K. (2006). Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic-overproducing Streptomyces coelicolor ribosomal rpsL mutant. Molecular Microbiology,61, 883–897.

    CAS  PubMed  Google Scholar 

  43. Lopatniuk, M., Myronovskyi, M., Nottebrock, A., Busche, T., Kalinowski, J., Ostash, B., et al. (2019). Effect of "ribosome engineering" on the transcription level and production of S. albus indigenous secondary metabolites. Applied Microbiology and Biotechnology,103, 7097–7110.

    CAS  PubMed  Google Scholar 

  44. Zhang, K., Mohsin, A., Dai, Y., Chen, Z., Zhuang, Y., Chu, J., et al. (2019). Combinatorial effect of ARTP mutagenesis and ribosome engineering on an industrial strain of Streptomyces albus S12 for enhanced biosynthesis of Salinomycin. Frontiers in Bioengineering and Biotechnology,7, 212.

    PubMed  PubMed Central  Google Scholar 

  45. Noren, C. J., Anthony-Cahill, S. J., Griffith, M. C., & Schultz, P. G. (1989). A general method for site-specific incorporation of unnatural amino acids into proteins. Science,244, 182–188.

    CAS  PubMed  Google Scholar 

  46. Chin, J. W. (2017). Expanding and reprogramming the genetic code. Nature,550, 53–60.

    CAS  PubMed  Google Scholar 

  47. Smolskaya, S., & Andreev, Y. A. (2019). Site-specific incorporation of unnatural amino acids into escherichia coli recombinant protein: methodology development and recent achievement. Biomolecules. https://doi.org/10.1111/eip.12846.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Chin, J. W. (2014). Expanding and reprogramming the genetic code of cells and animals. Annual Review of Biochemistry,83, 379–408.

    CAS  PubMed  Google Scholar 

  49. Wang, L., Brock, A., Herberich, B., & Schultz, P. G. (2001). Expanding the genetic code of Escherichia coli. Science,292, 498–500.

    CAS  PubMed  Google Scholar 

  50. Gao, W., Cho, E., Liu, Y., & Lu, Y. (2019). Advances and challenges in cell-free incorporation of unnatural amino acids into proteins. Frontiers in Pharmacology,10, 611.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Hoshika, S., Leal, N. A., Kim, M. J., Kim, M. S., Karalkar, N. B., Kim, H. J., et al. (2019). Hachimoji DNA and RNA: a genetic system with eight building blocks. Science,363, 884–887.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Magliery, T. J., Anderson, J. C., & Schultz, P. G. (2001). Expanding the genetic code: Selection of efficient suppressors of four-base codons and identification of "shifty" four-base codons with a library approach in Escherichia coli. Journal of Molecular Biology,307, 755–769.

    CAS  PubMed  Google Scholar 

  53. Suddala, K. C., & Zhang, J. (2019). High-affinity recognition of specific tRNAs by an mRNA anticodon-binding groove. Nature Structural & Molecular Biology,26, 1114–1122.

    CAS  Google Scholar 

  54. Battaglia, R. A., Grigg, J. C., & Ke, A. (2019). Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators. Nature Structural & Molecular Biology,26, 1106–1113.

    CAS  Google Scholar 

  55. Li, S., Su, Z., Lehmann, J., Stamatopoulou, V., Giarimoglou, N., Henderson, F. E., et al. (2019). Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions. Nature Structural & Molecular Biology,26, 1094–1105.

    CAS  Google Scholar 

  56. d'Aquino, A. E., Kim, D. S., & Jewett, M. C. (2018). Engineered ribosomes for basic science and synthetic biology. Annual Review of Chemical and Biomolecular Engineering,9, 311–340.

    CAS  PubMed  Google Scholar 

  57. Davis, L., & Chin, J. W. (2012). Designer proteins: applications of genetic code expansion in cell biology. Nature Reviews Molecular Cell Biology,13, 168–182.

    CAS  PubMed  Google Scholar 

  58. Lee, J., Schwieter, K. E., Watkins, A. M., Kim, D. S., Yu, H., Schwarz, K. J., et al. (2019). Expanding the limits of the second genetic code with ribozymes. Nature Communications,10, 5097.

    PubMed  PubMed Central  Google Scholar 

  59. Gan, R., Perez, J. G., Carlson, E. D., Ntai, I., Isaacs, F. J., Kelleher, N. L., et al. (2017). Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins. Biotechnology and Bioengineering,114, 1074–1086.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L., et al. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science,333, 348–353.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Cui, Z., Mureev, S., Polinkovsky, M. E., Tnimov, Z., Guo, Z., Durek, T., et al. (2017). Combining sense and nonsense codon reassignment for site-selective protein modification with unnatural amino acids. ACS Synthetic Biology,6, 535–544.

    CAS  PubMed  Google Scholar 

  62. Wang, L., & Schultz, P. G. (2004). Expanding the genetic code. Angewandte Chemie (International ed. in English),44, 34–66.

    Google Scholar 

  63. Cirino, P. C., Tang, Y., Takahashi, K., Tirrell, D. A., & Arnold, F. H. (2003). Global incorporation of norleucine in place of methionine in cytochrome P450 BM-3 heme domain increases peroxygenase activity. Biotechnology and Bioengineering,83, 729–734.

    CAS  PubMed  Google Scholar 

  64. Addy, P. S., Erickson, S. B., Italia, J. S., & Chatterjee, A. (2019). Labeling proteins at site-specifically incorporated 5-hydroxytryptophan residues using a chemoselective rapid azo-coupling reaction. Methods in Molecular Biology,2033, 239–251.

    CAS  PubMed  Google Scholar 

  65. Merten, H., Schaefer, J. V., Brandl, F., Zangemeister-Wittke, U., & Pluckthun, A. (2019). Facile site-specific multiconjugation strategies in recombinant proteins produced in Bacteria. Methods in Molecular Biology,2033, 253–273.

    CAS  PubMed  Google Scholar 

  66. Spicer, C. D., & Davis, B. G. (2014). Selective chemical protein modification. Nature Communications,5, 4740.

    CAS  PubMed  Google Scholar 

  67. Seo, M. H., Han, J., Jin, Z., Lee, D. W., Park, H. S., & Kim, H. S. (2011). Controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid. Analytical Chemistry,83, 2841–2845.

    CAS  PubMed  Google Scholar 

  68. Raliski, B. K., Howard, C. A., & Young, D. D. (2014). Site-specific protein immobilization using unnatural amino acids. Bioconjugate Chemistry,25, 1916–1920.

    CAS  PubMed  Google Scholar 

  69. Wu, J. C., Hutchings, C. H., Lindsay, M. J., Werner, C. J., & Bundy, B. C. (2015). Enhanced enzyme stability through site-directed covalent immobilization. Journal of Biotechnology,193, 83–90.

    CAS  PubMed  Google Scholar 

  70. Bednar, R. M., Golbek, T. W., Kean, K. M., Brown, W. J., Jana, S., Baio, J. E., et al. (2019). Immobilization of proteins with controlled load and orientation. ACS Applied Materials & Interfaces,11, 36391–36398.

    CAS  Google Scholar 

  71. Friedman, M., Ozer, E., Kushmaro, A., & Alfonta, L. (2019). Cellular localization of cytochrome bd in cyanobacteria using genetic code expansion. Biotechnology and Bioengineering,117, 523–530.

    Google Scholar 

  72. Albayrak, C., & Swartz, J. R. (2014). Direct polymerization of proteins. ACS Synthetic Biology,3, 353–362.

    CAS  PubMed  Google Scholar 

  73. Eiselt, E., Gonzalez, S., Martin, C., Chartier, M., Betti, C., Longpre, J. M., et al. (2019). Neurotensin analogues containing cyclic surrogates of tyrosine at position 11 improve NTS2 selectivity leading to analgesia without Hypotension and hypothermia. ACS Chemical Neuroscience,10, 4535–4544.

    CAS  PubMed  Google Scholar 

  74. Kelemen, R. E., Mukherjee, R., Cao, X., Erickson, S. B., Zheng, Y., & Chatterjee, A. (2016). A precise chemical strategy to alter the receptor specificity of the adeno-associated virus. Angewandte Chemie (International ed. in English),55, 10645–10649.

    CAS  Google Scholar 

  75. Huang, Y., & Liu, T. (2018). Therapeutic applications of genetic code expansion. Synthetic and Systems Biotechnology,3, 150–158.

    PubMed  PubMed Central  Google Scholar 

  76. Zhang, Z. W., Gildersleeve, J., Yang, Y. Y., Xu, R., Loo, J. A., Uryu, S., et al. (2004). A new strategy for the synthesis of glycoproteins. Science,303, 371–373.

    CAS  PubMed  Google Scholar 

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Acknowledgement

This publication was financially supported by the Nanhu Scholars Program for Young Scholars of XYNU, Startup Program, and Key Program for Natural Science Exploration of XYNU (2017-ZDYY-160), and Natural Science Foundation of China (U180410).

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  1. College of Life Sciences, and Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountains, Xinyang Normal University, Xinyang, 464000, China

    Tianwen Wang, Chen Liang, Yafei An, Sa Xiao, Hongjv Xu, Mengyuan Zheng, Lu Liu, Gaozhan Wang & Lei Nie

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  1. Tianwen Wang
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Wang, T., Liang, C., An, Y. et al. Engineering the Translational Machinery for Biotechnology Applications. Mol Biotechnol 62, 219–227 (2020). https://doi.org/10.1007/s12033-020-00246-y

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