Journal of Microbiology and Biotechnology

  • 제31권4호
  • /
  • Pages.570-583
  • /
  • 2021
  • /
  • 1017-7825(pISSN)
  • /
  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지
DOI QR코드

DOI QR Code

Enhanced Production of Soluble Pyrococcus furiosus α-Amylase in Bacillus subtilis through Chaperone Co-Expression, Heat Treatment and Fermentation Optimization

  • Zhang, Kang (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Tan, Ruiting (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Yao, Dongbang (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Su, Lingqia (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Xia, Yongmei (State Key Laboratory of Food Science and Technology, Jiangnan University) ;
  • Wu, Jing (State Key Laboratory of Food Science and Technology, Jiangnan University)
  • 투고 : 2021.02.01
  • 심사 : 2021.03.22
  • 발행 : 2021.04.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

키워드

참고문헌

  1. van der Maarel M, van der Veen B, Uitdehaag JCM, Leemhuis H, Dijkhuizen L. 2002. Properties and applications of starchconverting enzymes of the α-amylase family. J. Biotechnol. 94: 137-155. https://doi.org/10.1016/S0168-1656(01)00407-2
  2. Richardson TH, Tan X, Frey G, Callen W, Cabell M, Lam D, et al. 2002. A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable α-amylase. J. Biol. Chem. 277: 26501-26507. https://doi.org/10.1074/jbc.M203183200
  3. Khemakhem B, Ben Ali M, Aghajari N, Juy M, Haser R, Bejar S. 2009. Engineering of the α-amylase from Geobacillus stearothermophilus US100 for detergent incorporation. Biotechnol. Bioeng. 102: 380-389. https://doi.org/10.1002/bit.22083
  4. Saito N. 1973. A thermophilic extracellular α-amylase from Bacillus licheniformis. Arch. Biochem. Biophys. 155: 290-298. https://doi.org/10.1016/0003-9861(73)90117-3
  5. Yuuki T, Nomura T, Tezuka H, Tsuboi A, Yamagata H, Tsukagoshi N, et al. 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable α-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying α-amylases deduced from the DNA sequences. J. Biochem. 98: 1147-1156. https://doi.org/10.1093/oxfordjournals.jbchem.a135381
  6. Violet M, Meunier JC. 1989. Kinetic study of the irreversible thermal denaturation of Bacillus licheniformis α-amylase. Biochem. J. 263: 665-670. https://doi.org/10.1042/bj2630665
  7. Straub CT, Counts JA, Nguyen DMN, Wu CH, Zeldes BM, Crosby JR, et al. 2018. Biotechnology of extremely thermophilic archaea. FEMS Microbiol. Rev. 42: 543-578. https://doi.org/10.1093/femsre/fuy012
  8. Adams MW. 1993. Enzymes and proteins from organisms that grow near and above 100 degrees C. Annu. Rev. Microbiol. 47: 627-658. https://doi.org/10.1146/annurev.mi.47.100193.003211
  9. Demirjian DC, Moris-Varas F, Cassidy CS. 2001. Enzymes from extremophiles. Curr. Opin. Chem. Biol. 5: 144-151. https://doi.org/10.1016/S1367-5931(00)00183-6
  10. Laderman KA, Asada K, Uemori T, Mukai H, Taguchi Y, Kato I, et al. 1993. α-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus. Cloning and sequencing of the gene and expression in Escherichia coli. J. Biol. Chem. 268: 24402-24407. https://doi.org/10.1016/S0021-9258(20)80539-0
  11. Dong G, Vieille C, Savchenko A, Zeikus JG. 1997. Cloning, sequencing, and expression of the gene encoding extracellular α-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. Appl. Environ. Microbiol. 63: 3569-3576. https://doi.org/10.1128/aem.63.9.3569-3576.1997
  12. Shen W. 2003. Expression of α-amylase from Pyrococcus furiosus in different host cells. Doctor thesis. Jiangnan University.
  13. Wang P, Wang P, Tian J, Yu X, Chang M, Chu X, et al. 2016. A new strategy to express the extracellular α-amylase from Pyrococcus furiosus in Bacillus amyloliquefaciens. Sci. Rep. 6: 22229. https://doi.org/10.1038/srep22229
  14. Harwood CR, Cranenburgh R. 2008. Bacillus protein secretion: an unfolding story. Trends Microbiol. 16: 73-79. https://doi.org/10.1016/j.tim.2007.12.001
  15. Wei Y, Wang R, Du L, Lu J, Huang K, Huang R. 2005. Secreted expression of synthesized hyperthermophilic α-amylase gene pfa in Pichia pastoris. J. Chin. Biotechnol. 25: 65-69. https://doi.org/10.3969/j.issn.1671-8135.2005.01.014
  16. Horwich AL. 2013. Chaperonin-mediated protein folding. J. Biol. Chem. 288: 23622-23632. https://doi.org/10.1074/jbc.X113.497321
  17. Hartl FU, Hayer-Hartl M. 2002. Protein folding - Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295: 1852-1858. https://doi.org/10.1126/science.1068408
  18. Skjaerven L, Cuellar J, Martinez A, Valpuesta JM. 2015. Dynamics, flexibility, and allostery in molecular chaperonins. FEBS Lett. 589: 2522-2532. https://doi.org/10.1016/j.febslet.2015.06.019
  19. Zako T, Murase Y, Iizuka R, Yoshida T, Kanzaki T, Ide N, et al. 2006. Localization of prefoldin interaction sites in the hyperthermophilic group II chaperonin and correlations between binding rate and protein transfer rate. J. Mol. Biol. 364: 110-120. https://doi.org/10.1016/j.jmb.2006.08.088
  20. Martin-Benito J, Boskovic J, Gomez-Puertas P, Carrascosa JL, Simons CT, Lewis SA, et al. 2002. Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT. EMBO J. 21: 6377-6386. https://doi.org/10.1093/emboj/cdf640
  21. Whitehead TA, Boonyaratanakornkit BB, Hollrigl V, Clark DS. 2007. A filamentous molecular chaperone of the prefoldin family from the deep-sea hyperthermophile Methanocaldococcus jannaschii. Protein Sci. 16: 626-634. https://doi.org/10.1110/ps.062599907
  22. Glover DJ, Clark DS. 2015. Oligomeric assembly is required for chaperone activity of the filamentous γ-prefoldin. FEBS J. 282: 2985-2997. https://doi.org/10.1111/febs.13341
  23. Jakob RP, Koch JR, Burmann BM, Schmidpeter PA, Hunkeler M, Hiller S, et al. 2015. Dimeric structure of the bacterial extracellular foldase PrsA. J. Biol. Chem. 290: 3278-3292. https://doi.org/10.1074/jbc.M114.622910
  24. Ideno A, Yoshida T, Iida T, Furutani M, Maruyama T. 2001. FK506-binding protein of the hyperthermophilic archaeum, Thermococcus sp. KS-1, a cold-shock-inducible peptidyl-prolyl cis-trans isomerase with activities to trap and refold denatured proteins. Biochem. J. 357: 465-471. https://doi.org/10.1042/bj3570465
  25. Maruyama T, Suzuki R, Furutani M. 2004. Archaeal peptidyl prolyl cis-trans isomerases (PPIases) update 2004. Front. Biosci. 9: 1680-1720. https://doi.org/10.2741/1361
  26. Peng S, Chu Z, Lu J, Li D, Wang Y, Yang S, et al. 2016. Co-expression of chaperones from P. furiosus enhanced the soluble expression of the recombinant hyperthermophilic α-amylase in E. coli. Cell Stress Chaperon. 21: 477-484. https://doi.org/10.1007/s12192-016-0675-7
  27. Linden A, Niehaus F, Antranikian G. 2000. Single-step purification of a recombinant thermostable α-amylase after solubilization of the enzyme from insoluble aggregates. J. Chromatogr. B. 737: 253-259. https://doi.org/10.1016/S0378-4347(99)00364-3
  28. Wang L, Zhou Q, Chen H, Chu Z, Lu J, Zhang Y, et al. 2007. Efficient solubilization, purification of recombinant extracellular α-amylase from Pyrococcus furiosus expressed as inclusion bodies in Escherichia coli. J. Ind. Microbiol. Biotechnol. 34: 187-192. https://doi.org/10.1007/s10295-006-0185-1
  29. Su Y, Liu C, Fang H, Zhang D. 2020. Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microb. Cell Fact. 19: 173. https://doi.org/10.1186/s12934-020-01436-8
  30. Zhang K, Su L, Wu J. 2020. Recent advances in recombinant protein production by Bacillus subtilis. Annu. Rev. Food Sci. Technol. 11: 295-318. https://doi.org/10.1146/annurev-food-032519-051750
  31. Zhang K, Su L, Wu J. 2018. Enhanced extracellular pullulanase production in Bacillus subtilis using protease-deficient strains and optimal feeding. Appl. Microbiol. Biotechnol. 102: 5089-5103. https://doi.org/10.1007/s00253-018-8965-x
  32. Zhang K, Duan X, Wu J. 2016. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system. Sci. Rep-UK. 6: 27943. https://doi.org/10.1038/srep27943
  33. Wenzel M, Muller A, Siemann-Herzberg M, Altenbuchner J. 2011. Self-inducible Bacillus subtilis expression system for reliable and inexpensive protein production by high-cell-density fermentation. Appl. Environ. Microbiol. 77: 6419-6425. https://doi.org/10.1128/AEM.05219-11
  34. Anagnostopoulos C, Spizizen J. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81: 741-746. https://doi.org/10.1128/jb.81.5.741-746.1961
  35. Peters JM, Colavin A, Shi H, Czarny TL, Larson MH, Wong S, et al. 2016. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165: 1493-1506. https://doi.org/10.1016/j.cell.2016.05.003
  36. Westers H, Westers L, Darmon E, van Dijl JM, Quax WJ, Zanen G. 2006. The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis. FEBS J. 273: 3816-3827. https://doi.org/10.1111/j.1742-4658.2006.05389.x
  37. Caspers M, Brockmeier U, Degering C, Eggert T, Freudl R. 2010. Improvement of Sec-dependent secretion of a heterologous model protein in Bacillus subtilis by saturation mutagenesis of the N-domain of the AmyE signal peptide. Appl. Microbiol. Biotechnol. 86: 1877-1885. https://doi.org/10.1007/s00253-009-2405-x
  38. Freudl R. 2018. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb. Cell Fact. 17: 52. https://doi.org/10.1186/s12934-018-0901-3
  39. Ohtaki A, Kida H, Miyata Y, Ide N, Yonezawa A, Arakawa T, et al. 2008. Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins. J. Mol. Biol. 376: 1130-1141. https://doi.org/10.1016/j.jmb.2007.12.010
  40. Brown I, Dafforn TR, Fryer PJ, Cox PW. 2013. Kinetic study of the thermal denaturation of a hyperthermostable extracellular α-amylase from Pyrococcus furiosus. BBA-Proteins Proteom. 1834: 2600-2605. https://doi.org/10.1016/j.bbapap.2013.09.008
  41. Beadle BM, Baase WA, Wilson DB, Gilkes NR, Shoichet BK. 1999. Comparing the thermodynamic stabilities of a related thermophilic and mesophilic enzyme. Biochemistry 38: 2570-2576. https://doi.org/10.1021/bi9824902
  42. Smith JD, Richardson NE, Robinson AS. 2005. Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress. Biochim. Biophys. Acta 1752: 18-25. https://doi.org/10.1016/j.bbapap.2005.07.016
  43. Szilagyi A, Zavodszky P. 2000. Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey. Structure 8: 493-504. https://doi.org/10.1016/S0969-2126(00)00133-7
  44. Zhang K. 2018. Modification of Bacillus subtilis strain, promoter optimization and high-level expression of pullulanase. Doctor thesis. Jiangnan University.