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Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli

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Abstract

5-Aminolevulinic acid (ALA) is an important metabolic intermediate compound with high value and has recently been used in agriculture and medicine. In this study, we have constructed six recombinant Escherichia coli (E. coli) strains that are involved in pET system under the regulation of the T7 promoter and LacI to express codon-optimized hemA gene from Rhodobacter capsulatus (RchemA) for ALA production via the C4 pathway. Due to codon optimization, hemA has a high transcriptional level; however, most RcHemA proteins were formed as inclusion body. To improve expression in soluble form, the vector with TrxA fusion tag was successfully used and co-expressed with partner GroELS as chaperone in another vector. As a result, ALA production increased significantly from 1.21 to 3.67 g/L. In addition, optimal ALA production was developed through adjustment of induction time and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, as well as substrate addition conditions. By adopting a two-stage induction strategy, the highest ALA reached 5.66 g/L when 0.1 mM of IPTG was added at early exponential phase (i.e., OD600 was equal to 0.7 to 0.8), while 6 g/L of glycine, 2 g/L of succinate, and 0.03 mM of pyridoxal 5′-phosphate (PLP) were provided in the mid-exponential phase in fermentation.

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References

  1. Sasaki, K., Watanabe, M., & Tanaka, T. (2002). Biosynthesis, biotechnological production and applications of 5-aminolevulinic acid. Applied Microbiology and Biotechnology, 58(1), 23–29.

    Article  CAS  Google Scholar 

  2. Liu, S., Zhang, G., Li, X., & Zhang, J. (2014). Applied Microbiology and Biotechnology, 98(17), 7349–7357.

    Article  CAS  Google Scholar 

  3. Uehlinger, P., Zellweger, M., Wagnières, G., Juillerat-Jeanneret, L., et al. (2000). Journal of Photochemistry and Photobiology, B: Biology, 54(1), 72–80.

    Article  CAS  Google Scholar 

  4. Cornelius, J. F., Slotty, P. J., El Khatib, M., Giannakis, A., Senger, B., & Steiger, H. J. (2014). Photodiagnosis and Photodynamic Therapy, 11(1), 1–6.

    Article  CAS  Google Scholar 

  5. Noh, M. H., Lim, H. G., Park, S., Seo, S. W., & Jung, G. Y. (2017). Metabolic Engineering, 43, 1–8.

    Article  CAS  Google Scholar 

  6. Li, T., Guo, Y. Y., Qiao, G. Q., & Chen, G. Q. (2016). Microbial synthesis of 5-aminolevulinic acid and its coproduction with polyhydroxybutyrate. ACS Synthetic Biology, 5(11), 1264–1274.

    Article  CAS  Google Scholar 

  7. Shemin, D., & Russell, C. S. (1953). Journal of the American Chemical Society, 75(19), 4873–4874.

    Article  CAS  Google Scholar 

  8. Hunter, G. A., & Ferreira, G. C. (1999). Biochemistry, 38(12), 3711–3718.

    Article  CAS  Google Scholar 

  9. Ferreira, G. C., Neame, P. J., & Dailey, H. A. (1993). Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase. Protein Science, 2(11), 1959–1965.

    Article  CAS  Google Scholar 

  10. Shoolingin-Jordan, P. M., Al-Daihan, S., Alexeev, D., et al. (2003). Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1647(1–2), 361–366.

    Article  CAS  Google Scholar 

  11. Kang, Z., Wang, Y., Gu, P., Wang, Q., et al. (2011). Metabolic Engineering, 13(5), 492–498 21.

    Article  CAS  Google Scholar 

  12. Schauer, S., Chaturvedi, S., Randau, L., Moser, J., Kitabatake, M., Lorenz, S., Verkamp, E., Schubert, W. D., Nakayashiki, T., Murai, M., Wall, K., Thomann, H. U., Heinz, D. W., Inokuchi, H., Söll, D., & Jahn, D. (2002). Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate. Journal of Biological Chemistry, 277(50), 48657–48663.

    Article  CAS  Google Scholar 

  13. Chung, S. Y., Seo, K. H., & Rhee, J. I. (2005). Process Biochemistry, 40(1), 385–394 26.

    Article  CAS  Google Scholar 

  14. Liu, X. X., Wang, L., Wang, Y. J., & Cai, L. L. (2010). D-glucose enhanced 5-aminolevulinic acid production in recombinant Escherichia coli culture. Applied Biochemistry and Biotechnology, 160(3), 822–830.

    Article  CAS  Google Scholar 

  15. Zhang, J., Kang, Z., Ding, W., Chen, J., & du, G. (2016). Integrated optimization of the in vivo heme biosynthesis pathway and the in vitro iron concentration for 5-aminolevulinate production. Applied Biochemistry and Biotechnology, 178(6), 1252–1262.

    Article  CAS  Google Scholar 

  16. Yasukawa, T., Kanei-Ishii, C., Maekawa, T., Fujimoto, J., Yamamoto, T., & Ishii, S. (1995). Increase of solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin. Journal of Biological Chemistry, 270(43), 25328–25331.

    Article  CAS  Google Scholar 

  17. Quan, S., & Bardwell, J. C. (2012). Chaperone discovery. Bioessays, 34(11), 973–981.

    Article  CAS  Google Scholar 

  18. Lou, J. W., Zhu, L., Wu, M. B., Yang, L. R. Lin, J. P., &, Cen, P. L. (2014) Journal of Zhejiang University. Science. B 15(5), 491–499.

  19. Fanica-Gaignier, M., & Clement-Metral, J. (1973). 5-AMINOLEVULINIC-acid synthetase of Rhodopseudomonas spheroides Y. Purification and some properties. European Journal of Biochemistry, 40(1), 13–18.

    Article  CAS  Google Scholar 

  20. Yubisui, T., & Yoneyama, Y. (1972). Aminolevulinic acid synthetase of Rhodopseudomonas spheroides: purification and properties of the enzyme. Archives of Biochemistry and Biophysics, 150(1), 77–85.

    Article  CAS  Google Scholar 

  21. Jordan, P. M., & Laghai-Newton, A. (1986). Methods in enzymology, vol. 123 (pp. 435–443). Academic Press.

  22. Nakakuki, M., Yamauchi, K., Hayashi, N., & Kikuchi, G. (1980). Purification and some properties of delta-aminolevulinate synthase from the rat liver cytosol fraction and immunochemical identity of the cytosolic enzyme and the mitochondrial enzyme. Journal of Biological Chemistry, 255(4), 1738–1745.

    CAS  PubMed  Google Scholar 

  23. Volland, C., & Felix, F. (1984). Isolation and properties of 5-aminolevulinate synthase from the yeast Saccharomyces cerevisiae. European Journal of Biochemistry, 142(3), 551–557.

    Article  CAS  Google Scholar 

  24. Ferreira, G. C., & Gong, J. (1995). 5-Aminolevulinate synthase and the first step of heme biosynthesis. Journal of Bioenergetics and Biomembranes, 27(2), 151–159.

    Article  CAS  Google Scholar 

  25. Stojanovski, B. M., & Ferreira, G. C. (2015). Murine erythroid 5-aminolevulinate synthase: adenosyl-binding site Lys221 modulates substrate binding and catalysis. FEBS Open Bio, 5, 824–831.

    Article  CAS  Google Scholar 

  26. Zhu, C., Chen, J., Wang, Y., Wang, L., Guo, X., Chen, N., Zheng, P., Sun, J., & Ma, Y. (2019). Enhancing 5-aminolevulinic acid tolerance and production by engineering the antioxidant defense system of Escherichia coli. Biotechnology and Bioengineering, 116(8), 2018–2028.

    Article  CAS  Google Scholar 

  27. Hayer-Hartl, M., Bracher, A., & Hartl, F. U. (2016). Trends in Biochemical Sciences, 41(1), 62–76.

    Article  CAS  Google Scholar 

  28. Xu, Z., Horwich, A. L., & Sigler, P. B. (1997). Nature, 388(6644), 741.

    Article  CAS  Google Scholar 

  29. Collet, J. F., & Messens, J. (2010). Antioxidants & Redox Signaling, 13(8), 1205–1216.

    Article  CAS  Google Scholar 

  30. Hayer-Hartl, M., Bracher, A., & Hartl, F. U. (2016). The GroEL-GroES chaperonin machine: a nano-cage for protein folding. Trends in Biochemical Sciences, 41(1), 62–76.

    Article  CAS  Google Scholar 

  31. Fu, W., Lin, J., & Cen, P. (2010). Applied Biochemistry and Biotechnology, 160(2), 456–466.

    Article  CAS  Google Scholar 

  32. Su, T., Guo, Q., Zheng, Y., Liang, Q., et al. (2019). Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2019.01731.

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Funding

This work received financial support from the Ministry of Science and Technology (MOST 108-2221-E-006-004-MY3, MOST 108-2218-E-006-006, and MOST-108-2621-M-006-015) in Taiwan.

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  1. Department of Chemical Engineering, National Cheng Kung University, Tainan, 701, Taiwan

    Tzu-Hsuan Yu, Ying-Chen Yi, I-Tai Shih & I-Son Ng

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  1. Tzu-Hsuan Yu
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  2. Ying-Chen Yi
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Correspondence to I-Son Ng.

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Yu, TH., Yi, YC., Shih, IT. et al. Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli. Appl Biochem Biotechnol 191, 299–312 (2020). https://doi.org/10.1007/s12010-019-03178-9

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