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Efficient Expression of Human Lysozyme Through the Increased Gene Dosage and Co-expression of Transcription Factor Hac1p in Pichia pastoris

Abstract

In this work, the high-level expression of the human lysozyme (HLY) was achieved by both optimization of the gene copy number and co-expression of the transcription factor Hac1p for the unfolded protein response (UPR) in the host strain Pichia pastoris KM71H. A series of recombinant constructs with various numbers of HLY expression cassettes was generated for the production of recombinant strains integrated with different copies of the HLY gene. The copy number of the HLY gene was determined by real-time quantitative polymerase chain reaction, and the recombinant strains of P. pastoris carrying one, two, three, four, or six copies of the HLY gene were obtained. Maximum extracellular protein and lysozyme enzyme activity reached 436.99 ± 26.08 μg/mL and 61,900 ± 2036.47 U/mL, respectively, in the recombinant strain HLYH4-3 with the four copies of the HLY gene after shaking flask fermentation. Moreover, the co-expression of the transcription factor Hac1p in the recombinant strains further enhanced the HLY yields. Extracellular protein and lysozyme enzyme activity, respectively, reached 517.82 ± 4.19 μg/mL and 78,600 ± 1134.95 U/mL by using the Hac1p co-expression strain HLYH4-3/Hac1p. These values are the highest recorded level of human lysozyme expressed by P. pastoris in shaking flask fermentation so far.

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References

  1. 1.

    Callewaert L, Michiels CW (2010) Lysozymes in the animal kingdom. J Biosci 35(1):127–160. https://doi.org/10.1007/s12038-010-0015-5

  2. 2.

    Van Herreweghe JM, Michiels CW (2012) Invertebrate lysozymes: diversity and distribution, molecular mechanism and in vivo function. J Biosci 37(2):327–348. https://doi.org/10.1007/s12038-012-9201-y

  3. 3.

    Shahmohammadi A (2018) Lysozyme separation from chicken egg white: a review. Eur Food Res Technol 244(4):577–593. https://doi.org/10.1007/s00217-017-2993-0

  4. 4.

    Syngai GG, Ahmed G (2019) Chapter 11—lysozyme: a natural antimicrobial enzyme of interest in food applications. In: Kuddus M (ed) Enzymes in food biotechnology. Academic Press, London, pp 169–179. https://doi.org/10.1016/B978-0-12-813280-7.00011-6

  5. 5.

    Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12):5301–5317. https://doi.org/10.1007/s00253-014-5732-5

  6. 6.

    Goda S, Takano K, Yamagata Y, Katakura Y, Yutani K (2000) Effect of extra N-terminal residues on the stability and folding of human lysozyme expressed in Pichia pastoris. Protein Eng 13(4):299–307. https://doi.org/10.1093/protein/13.4.299

  7. 7.

    Xiong R, Chen J, Chen J (2008) Secreted expression of human lysozyme in the yeast Pichia pastoris under the direction of the signal peptide from human serum albumin. Biotechnol Appl Biochem 51(3):129–134. https://doi.org/10.1042/BA20070205

  8. 8.

    Wei J-T, Tang C-D, Wu M-C, Liu G-L, Shi H-L, Li J-F (2012) Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris. Mol Med Rep 6(1):173–178. https://doi.org/10.3892/mmr.2012.878

  9. 9.

    Yu Y, Zhou X, Wu S, Wei T, Yu L (2014) High-yield production of the human lysozyme by Pichia pastoris SMD1168 using response surface methodology and high-cell-density fermentation. Electron J Biotechnol 17(6):311–316. https://doi.org/10.1016/j.ejbt.2014.09.006

  10. 10.

    Sha C, Yu X-W, Li F, Xu Y (2013) Impact of gene dosage on the production of lipase from Rhizopus chinensis CCTCC M201021 in Pichia pastoris. Appl Biochem Biotechnol 169(4):1160–1172. https://doi.org/10.1007/s12010-012-0050-9

  11. 11.

    He M, Wu D, Wu J, Chen J (2014) Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production. J Ind Microbiol Biotechnol 41(1):105–114. https://doi.org/10.1007/s10295-013-1341-z

  12. 12.

    Massahi A, Çalık P (2016) Endogenous signal peptides in recombinant protein production by Pichia pastoris: from in-silico analysis to fermentation. J Theor Biol 408:22–33. https://doi.org/10.1016/j.jtbi.2016.07.039

  13. 13.

    Lin-Cereghino GP, Stark CM, Kim D, Chang J, Shaheen N, Poerwanto H, Agari K, Moua P, Low LK, Tran N, Huang AD, Nattestad M, Oshiro KT, Chang JW, Chavan A, Tsai JW, Lin-Cereghino J (2013) The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene 519(2):311–317. https://doi.org/10.1016/j.gene.2013.01.062

  14. 14.

    Yang J, Lu Z, Chen J, Chu P, Cheng Q, Liu J, Ming F, Huang C, Xiao A, Cai H, Zhang L (2016) Effect of cooperation of chaperones and gene dosage on the expression of porcine PGLYRP-1 in Pichia pastoris. Appl Microbiol Biotechnol 100(12):5453–5465. https://doi.org/10.1007/s00253-016-7372-4

  15. 15.

    Ruth C, Buchetics M, Vidimce V, Kotz D, Naschberger S, Mattanovich D, Pichler H, Gasser B (2014) Pichia pastoris Aft1—a novel transcription factor, enhancing recombinant protein secretion. Microb Cell Fact 13(1):120. https://doi.org/10.1186/s12934-014-0120-5

  16. 16.

    Bankefa OE, Wang M, Zhu T, Li Y (2018) Hac1p homologues from higher eukaryotes can improve the secretion of heterologous proteins in the yeast Pichia pastoris. Biotech Lett 40(7):1149–1156. https://doi.org/10.1007/s10529-018-2571-y

  17. 17.

    Marsalek L, Gruber C, Altmann F, Aleschko M, Mattanovich D, Gasser B, Puxbaum V (2017) Disruption of genes involved in CORVET complex leads to enhanced secretion of heterologous carboxylesterase only in protease deficient Pichia pastoris. Biotechnol J 12(5):1600584. https://doi.org/10.1002/biot.201600584

  18. 18.

    Wu D, Hao Y-Y, Chu J, Zhuang Y-P, Zhang S-L (2008) Inhibition of degradation and aggregation of recombinant human consensus interferon-α mutant expressed in Pichia pastoris with complex medium in bioreactor. Appl Microbiol Biotechnol 80(6):1063. https://doi.org/10.1007/s00253-008-1629-5

  19. 19.

    Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85(4):367–375. https://doi.org/10.1002/bit.10904

  20. 20.

    Zhu T, Guo M, Zhuang Y, Chu J, Zhang S (2011) Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes. Appl Microbiol Biotechnol 89(4):1127–1135. https://doi.org/10.1007/s00253-010-2944-1

  21. 21.

    Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, Callewaert N (2010) The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Fact 9(1):49. https://doi.org/10.1186/1475-2859-9-49

  22. 22.

    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45–e45. https://doi.org/10.1093/nar/29.9.e45

  23. 23.

    Olmo R, Teijón C, Muñiz E, Beneit JV, Villarino AL, Blanco MD (2012) Modulation of lysozyme activity by lead administered by different routes. In vitro study and analysis in blood, kidney, and lung. Biol Trace Elem Res 149(3):405–411. https://doi.org/10.1007/s12011-012-9446-1

  24. 24.

    Yu M, Wen S, Tan T (2010) Enhancing production of Yarrowia lipolytica lipase Lip2 in Pichia pastoris. Eng Life Sci 10(5):458–464

  25. 25.

    Yang H, Zhai C, Yu X, Li Z, Tang W, Liu Y, Ma X, Zhong X, Li G, Wu D, Ma L (2016) High-level expression of proteinase K from Tritirachium album Limber in Pichia pastoris using multi-copy expression strains. Protein Expr Purif 122:38–44. https://doi.org/10.1016/j.pep.2016.02.006

  26. 26.

    Zhu T, Guo M, Tang Z, Zhang M, Zhuang Y, Chu J, Zhang S (2009) Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris. J Appl Microbiol 107(3):954–963. https://doi.org/10.1111/j.1365-2672.2009.04279.x

  27. 27.

    Cos O, Serrano A, Montesinos JL, Ferrer P, Cregg JM, Valero F (2005) Combined effect of the methanol utilization (Mut) phenotype and gene dosage on recombinant protein production in Pichia pastoris fed-batch cultures. J Biotechnol 116(4):321–335. https://doi.org/10.1016/j.jbiotec.2004.12.010

  28. 28.

    Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101(3):249–258. https://doi.org/10.1016/S0092-8674(00)80835-1

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Acknowledgements

This study was financially supported by the Open Project of Hubei Key Laboratory of Animal Nutrition and Feed Science (Grant Number 201809).

Funding

This study was financially supported by the Open Project of Hubei Key Laboratory of Animal Nutrition and Feed Science (Grant Number 201809).

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Correspondence to Dazhong Yan.

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Liu, J., Han, Q., Cheng, Q. et al. Efficient Expression of Human Lysozyme Through the Increased Gene Dosage and Co-expression of Transcription Factor Hac1p in Pichia pastoris. Curr Microbiol (2020). https://doi.org/10.1007/s00284-019-01872-9

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