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Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution-guided mutagenesis

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Abstract

Streptomyces griseus trypsin (SGT) is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions. However, SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue. In this study, by engineering the α signal peptide in Pichia pastoris, we increased the SGT amidase activity to 67.91 U mL−1 in shake flask cultures. Afterwards, we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S, V177I and E180M with increased catalytic efficiencies. On this basis, we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL−1 in shake flasks and 2506 U mL−1 in 3-L fed-batch cultures. Moreover, single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5, which was 2 times better than the animal-sourced trypsin.

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All data generated or analyzed during this study are included in this article and e-supplementary data for this work can be found in e-version of this paper online.

References

  1. Carter P, Wells JA. Dissecting the catalytic triad of a serine protease. Nature. 1988;332(6164):564–8. https://doi.org/10.1038/332564a0.

    Article  CAS  PubMed  Google Scholar 

  2. Kraut J. Serine proteases: structure and mechanism of catalysis. Annu Rev Biochem. 1977;46:331–58. https://doi.org/10.1146/annurev.bi.46.070177.001555.

    Article  CAS  PubMed  Google Scholar 

  3. Williams JA. Trypsin. In: Johnson LR, editor. Encyclopedia of gastroenterology. New York: Elsevier; 2004. p. 533–4.

    Chapter  Google Scholar 

  4. Mao Y, Krischke M, Hengst C, Kulozik U. Comparison of the influence of pH on the selectivity of free and immobilized trypsin for β-lactoglobulin hydrolysis. Food Chem. 2018;253:194–202. https://doi.org/10.1016/j.foodchem.2018.01.151.

    Article  CAS  PubMed  Google Scholar 

  5. Ketnawa S, Benjakul S, Martínez-Alvarez O, Rawdkuen S. Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: bioactivity and stability. Food Chem. 2017;215:383–90. https://doi.org/10.1016/j.foodchem.2016.07.145.

    Article  CAS  PubMed  Google Scholar 

  6. Gudmundsdóttir Á, Hilmarsson H, Stefansson B. Potential use of atlantic cod trypsin in biomedicine. Biomed Res Int. 2013;2013: 749078. https://doi.org/10.1155/2013/749078.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Liu H, Zhou X, Tian S, Hao X, You J, Zhang Y. Two-step transpeptidation of the insulin precursor expressed in Pichia pastoris to insulin ester via trypsin-catalyzed cleavage and coupling. Biotechnol Appl Biochem. 2014;61(4):408–17. https://doi.org/10.1002/bab.1186.

    Article  CAS  PubMed  Google Scholar 

  8. Lombardi J, Woitovich Valetti N, Picó G, Boeris V. Obtainment of a highly concentrated pancreatic serine proteases extract from bovine pancreas by precipitation with polyacrylate. Sep Purif Technol. 2013;116:170–4. https://doi.org/10.1016/j.seppur.2013.05.047.

    Article  CAS  Google Scholar 

  9. Grishina Z, Ostrowska E, Halangk W, Sahin-Tóth M, Reiser G. Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1. Br J Pharmacol. 2005;146(7):990–9. https://doi.org/10.1038/sj.bjp.0706410.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. EFSA Panel on Food Contact Materials E, Aids P, Lambré C, Barat Baviera JM, Bolognesi C, Cocconcelli PS, et al. Safety evaluation of the food enzyme trypsin from porcine pancreas. EFSA J. 2022;20(1):e07008. https://doi.org/10.2903/j.efsa.2022.7008.

    Article  CAS  Google Scholar 

  11. Zhang Y, Liang Q, Zhang C, Zhang J, Du G, Kang Z. Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor. Microb Cell Fact. 2020;19(1):88. https://doi.org/10.1186/s12934-020-01338-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Walmsley SJ, Rudnick PA, Liang Y, Dong Q, Stein SE, Nesvizhskii AI. Comprehensive analysis of protein digestion using six trypsins reveals the origin of trypsin as a significant source of variability in proteomics. J Proteome Res. 2013;12(12):5666–80. https://doi.org/10.1021/pr400611h.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Page MJ, Wong S-L, Hewitt J, Strynadka NCJ, MacGillivray RTA. Engineering the primary substrate specificity of Streptomyces griseus trypsin. Biochemistry. 2003;42(30):9060–6. https://doi.org/10.1021/bi0344230.

    Article  CAS  PubMed  Google Scholar 

  14. Oh EA, Kim M-S, Chi W-J, Kim J-H, Hong S-K. Characterization of the sgtR1 and sgtR2 genes and their role in regulating expression of the sprT gene encoding Streptomyces griseus trypsin. FEMS Microbiol Lett. 2007;276(1):75–82. https://doi.org/10.1111/j.1574-6968.2007.00907.x.

    Article  CAS  PubMed  Google Scholar 

  15. Ling Z, Ma T, Li J, Du G, Kang Z, Chen J. Functional expression of trypsin from Streptomyces griseus by Pichia pastoris. J Ind Microbiol Biotechnol. 2012;39(11):1651–62. https://doi.org/10.1007/s10295-012-1172-3.

    Article  CAS  PubMed  Google Scholar 

  16. Ling Z, Ma T, Li J, Du G, Kang Z, Chen J. Functional expression of trypsin from Streptomyces griseus by Pichia pastoris. J Ind Microbiol Biotechnol. 2012;39:1651–62. https://doi.org/10.1007/s10295-012-1172-3.

    Article  CAS  PubMed  Google Scholar 

  17. Ling Z, Liu Y, Teng S, Kang Z, Zhang J, Chen J, et al. Rational design of a novel propeptide for improving active production of Streptomyces griseus Trypsin in Pichia pastoris. Appl Environ Microbiol. 2013;79(12):3851–5. https://doi.org/10.1128/AEM.00376-13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang Y, Ling Z, Du G, Chen J, Kang Z. Improved production of active Streptomyces griseus trypsin with a novel auto-catalyzed strategy. Sci Rep. 2016;6(1):23158. https://doi.org/10.1038/srep23158.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Tamura K, Stecher G, Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol Biol Evol. 2021;38(7):3022–7. https://doi.org/10.1093/molbev/msab120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wang Z, Wang Y, Zhang D, Li J, Hua Z, Du G, et al. Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation. Biores Technol. 2010;101(4):1318–23. https://doi.org/10.1016/j.biortech.2009.09.025.

    Article  CAS  Google Scholar 

  21. Zhang Y, Huang H, Yao X, Du G, Chen J, Kang Z. High-yield secretory production of stable, active trypsin through engineering of the N-terminal peptide and self-degradation sites in Pichia pastoris. Bioresour Technol. 2018;247:81–7. https://doi.org/10.1016/j.biortech.2017.08.006.

    Article  CAS  PubMed  Google Scholar 

  22. Ben Azoun S, Belhaj AE, Gongrich R, Gasser B, Kallel H. Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris. Microb Biotechnol. 2016;9(3):355–68. https://doi.org/10.1111/1751-7915.12350.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018;46(W1):W296–303. https://doi.org/10.1093/nar/gky427.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kleffner R, Flatten J, Leaver-Fay A, Baker D, Siegel JB, Khatib F, et al. Foldit standalone: a video game-derived protein structure manipulation interface using Rosetta. Bioinformatics. 2017;33(17):2765–7. https://doi.org/10.1093/bioinformatics/btx283.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31(2):455–61. https://doi.org/10.1002/jcc.21334.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lin-Cereghino GP, Stark CM, Kim D, Chang J, Shaheen N, Poerwanto H, et al. The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene. 2013;519(2):311–7. https://doi.org/10.1016/j.gene.2013.01.062.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chahal S, Wei P, Moua P, Park SPJ, Kwon J, Patel A, et al. Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris. Gene. 2017;598:50–62. https://doi.org/10.1016/j.gene.2016.10.040.

    Article  CAS  PubMed  Google Scholar 

  28. Huang C Jr, Damasceno LM, Anderson KA, Zhang S, Old LJ, Batt CA. A proteomic analysis of the Pichia pastoris secretome in methanol-induced cultures. Appl Microbiol Biotechnol. 2011;90(1):235–47. https://doi.org/10.1007/s00253-011-3118-5.

    Article  CAS  PubMed  Google Scholar 

  29. Liang Q, Shi J, Jin X, Du G, Kang Z. Optimization of enterokinase secretion in Pichia pastoris. Sheng Wu Gong Cheng Xue Bao. 2020;36(8):1689–98. https://doi.org/10.13345/j.cjb.190577.

    Article  CAS  PubMed  Google Scholar 

  30. Bae J-H, Sung BH, Seo J-W, Kim CH, Sohn J-H. A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2016;100(24):10453–61. https://doi.org/10.1007/s00253-016-7722-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Jiang N, Xu C, Zhang L, Chen J. “Resurrected” human-source urate oxidase with high uricolytic activity and stability. Enzyme Microb Technol. 2021;149: 109852. https://doi.org/10.1016/j.enzmictec.2021.109852.

    Article  CAS  PubMed  Google Scholar 

  32. Stolterfoht H, Steinkellner G, Schwendenwein D, Pavkov-Keller T, Gruber K, Winkler M. Identification of key residues for enzymatic carboxylate reduction. Front Microbiol. 2018;9:250. https://doi.org/10.3389/fmicb.2018.00250.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Spilliaert R, Gudmundsdóttir Á. Atlantic cod trypsin Y—member of a novel trypsin group. Mar Biotechnol. 1999;1(6):598–607. https://doi.org/10.1007/PL00011815.

    Article  CAS  Google Scholar 

  34. Evnin L, Vásquez J, Craik C. Substrate specificity of trypsin investigated by using a gentic selection. Proc Natl Acad Sci USA. 1990;87:6659–63. https://doi.org/10.1073/pnas.87.17.6659.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Weiner SJ, Seibel GL, Kollman PA. The nature of enzyme catalysis in trypsin. Proc Natl Acad Sci. 1986;83(3):649. https://doi.org/10.1073/pnas.83.3.649.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Reetz MT, Carballeira JD. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc. 2007;2(4):891–903. https://doi.org/10.1038/nprot.2007.72.

    Article  CAS  PubMed  Google Scholar 

  37. Liao X, Zhao J, Liang S, Jin J, Li C, Xiao R, et al. Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris. Biotechnol Biofuels. 2019;12:38. https://doi.org/10.1186/s13068-019-1377-z.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Burgard J, Grünwald-Gruber C, Altmann F, Zanghellini J, Valli M, Mattanovich D, et al. The secretome of Pichia pastoris in fed-batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time. Microb Biotechnol. 2020;13(2):479–94. https://doi.org/10.1111/1751-7915.13499.

    Article  CAS  PubMed  Google Scholar 

  39. Maiolo M, Zhang X, Gil M, Anisimova M. Progressive multiple sequence alignment with indel evolution. BMC Bioinform. 2018;19(1):331. https://doi.org/10.1186/s12859-018-2357-1.

    Article  Google Scholar 

  40. Labas YA, Gurskaya NG, Yanushevich YG, Fradkov AF, Lukyanov KA, Lukyanov SA, et al. Diversity and evolution of the green fluorescent protein family. Proc Natl Acad Sci. 2002;99(7):4256. https://doi.org/10.1073/pnas.062552299.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was financially supported by the Jiangsu Province Natural Science Fund for Distinguished Young Scholars (BK20200025), a grant from the Key Technologies R & D Program of Jiangsu Province (BE2019630), the China Postdoctoral Science Foundation (2021M691286).

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Authors and Affiliations

  1. The Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China

    Jingcheng Shi, Chaofan Duan, Bo Pang, Yang Wang, Guocheng Du & Zhen Kang

  2. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China

    Jingcheng Shi, Chaofan Duan, Bo Pang, Yang Wang & Zhen Kang

  3. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Guocheng Du

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  1. Jingcheng Shi
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Correspondence to Zhen Kang.

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Shi, J., Duan, C., Pang, B. et al. Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution-guided mutagenesis. Syst Microbiol and Biomanuf 3, 287–297 (2023). https://doi.org/10.1007/s43393-022-00107-6

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