Skip to main content
SpringerLink
Log in

Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris

  • Biocatalysis - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

A hydroxynitrile lyase (HNL) from the millipede Chamberlinius hualienensis has high potential for industrial use in the synthesis of cyanohydrins. However, obtaining sufficient amounts of millipedes is difficult, and the production of the Chamberlinius hualienensis HNL (ChuaHNL) in E. coli has not been very successful. Therefore, we investigated the conditions required for high-yield heterologous production of this enzyme using Pichia pastoris. When we employed P. pastoris to express His-ChuaHNL, the yield was very low (22.6 ± 3.8 U/L culture). Hence, we investigated the effects of ChuaHNL codon optimization and the co-production of two protein disulfide isomerases (PDIs) [from P. pastoris (PpPDI) and C. hualienensis (ChuaPDI1, ChuaPDI2)] on His-ChuaHNL production. The productivity of His-ChuaHNL was increased approximately 140 times per unit culture to 3170 ± 144.7 U/L by the co-expression of codon-optimized ChuaHNL and PpPDI. Moreover, we revealed that the N-glycosylation on ChuaHNL had a large effect on the stability, enzyme secretion, and catalytic properties of ChuaHNL in P. pastoris. This study demonstrates an economical and efficient approach for the production of HNL, and the data show that glycosylation has a large effect on the enzyme properties and the P. pastoris expression system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Asano Y, Tamura K, Doi N, Ueatrongchit T, H-Kittikun A, Ohmiya T (2005) Screening for new hydroxynitrilases from plants. Biosci Biotechnol Biochem 69:2349–2357. https://doi.org/10.1271/bbb.69.2349

    Article  CAS  PubMed  Google Scholar 

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  3. Caruso CS, de Fatima Travensolo R, de Campus Bicudo R, de Macedo Lemos EG, Ulian de Araujo AP, Carrilho E (2009) alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: cloning, expression, and characterization. Microb Pathog 47:118–127. https://doi.org/10.1016/j.micpath.2009.06.007

    Article  CAS  PubMed  Google Scholar 

  4. Chang S-W, Lee G-C, Shaw J-F (2006) Codon optimization of Candida rugosa lip1 gene for improving expression in Pichia pastoris and biochemical characterization of the purified recombinant LIP1 lipase. J Agric Food Chem 54:815–822. https://doi.org/10.1021/jf052183k

    Article  CAS  PubMed  Google Scholar 

  5. Dadashipour M, Asano Y (2011) Hydroxynitrile lyases: insights into biochemistry, discovery, and engineering. ACS Catal 1:1121–1149. https://doi.org/10.1021/cs200325q

    Article  CAS  Google Scholar 

  6. Dadashipour M, Ishida Y, Yamamoto K, Asano Y (2015) Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis. Proc Natl Acad Sci USA 112:10605–10610

    Article  CAS  PubMed  Google Scholar 

  7. Fukuta Y, Nanda S, Kato Y, Yurimoto H, Sakai Y, Komeda H, Asano Y (2011) Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris. Biosci Biotechnol Biochem 75:214–220. https://doi.org/10.1271/bbb.100187

    Article  CAS  PubMed  Google Scholar 

  8. Gao Z, Li Z, Zhang Y, Huang H, Li M, Zhou L, Tang Y, Yao B, Zhang W (2012) High-level expression of the Penicillium notatum glucose oxidase gene in Pichia pastoris using codon optimization. Biotechnol Lett 34:507–514. https://doi.org/10.1007/s10529-011-0790-6

    Article  CAS  PubMed  Google Scholar 

  9. Hu S, Li L, Qiao J, Guo Y, Cheng L, Liu J (2006) Codon optimization, expression, and characterization of an internalizing anti-ErbB2 single-chain antibody in Pichia pastoris. Protein Expr Purif 47:249–257

    Article  CAS  PubMed  Google Scholar 

  10. Hussain Z, Wiedner R, Steiner K, Hajek T, Avi M, Hecher B, Sessitsch A, Schwab H (2012) Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins. Appl Environ Microbiol 78:2053–2055. https://doi.org/10.1128/AEM.06899-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ishida Y, Kuwahara Y, Dadashipour M, Ina A, Yamaguchi T, Morita M, Ichiki Y, Asano Y (2016) A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase. Sci Rep 6:26998. https://doi.org/10.1038/srep26998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  13. Nuylert A, Ishida Y, Asano Y (2017) Effect of glycosylation on the biocatalytic properties of hydroxynitrile lyase from the passion fruit, Passiflora edulis: a comparison of natural and recombinant enzymes. ChemBioChem 18:257–265. https://doi.org/10.1002/cbic.201600447

    Article  CAS  PubMed  Google Scholar 

  14. Osbourn AE (1996) Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821–1831. https://doi.org/10.1105/tpc.8.10.1821

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sinclair G, Choy FY (2002) Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif 26:96–105

    Article  CAS  PubMed  Google Scholar 

  16. Tian G, Xiang S, Noiva R, Lennarz WJ, Schindelin H (2006) The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. Cell 124:61–73

    Article  CAS  PubMed  Google Scholar 

  17. Wajant H, Effenberger F (1996) Hydroxynitrile lyases of higher plants. Biol Chem 377:611–617

    CAS  PubMed  Google Scholar 

  18. Weis R, Gaisberger R, Gruber K, Glieder A (2007) Serine scanning: a tool to prove the consequences of N-glycosylation of proteins. J Biotechnol 129:50–61. https://doi.org/10.1016/j.jbiotec.2006.12.001

    Article  CAS  PubMed  Google Scholar 

  19. Wiedner R, Gruber-Khadjawi M, Schwab H, Steiner K (2014) Discovery of a novel (R)-selective bacterial hydroxynitrile lyase from Acidobacterium capsulatum. Comput Struct Biotechnol J 10:58–62. https://doi.org/10.1016/j.csbj.2014.07.002

    Article  PubMed  PubMed Central  Google Scholar 

  20. Wildt S, Gerngross TU (2005) The humanization of N-glycosylation pathways in yeast. Nat Rev Micro 3:119–128. https://doi.org/10.1038/nrmicro1087

    Article  CAS  Google Scholar 

  21. Yamaguchi T, Nuylert A, Ina A, Tanabe T, Asano Y (2018) Hydroxynitrile lyases from cyanogenic millipedes: molecular cloning, heterologous expression, and whole-cell biocatalysis for the production of (R)-mandelonitrile. Sci Rep 8:3051. https://doi.org/10.1038/s41598-018-20190-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhao GJ, Yang ZQ, Guo YH (2011) Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris. J Biosci Bioeng 112:321–325. https://doi.org/10.1016/j.jbiosc.2011.06.015

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Yuko Ishida for analyzing the RNA sequence of C. hualienensis. This work was supported by ERATO (Exploratory Research for Advanced Technology Program), Asano Active Enzyme Molecule Project of Japan Science and Technology Agency (Grant No. JPMJER1102). This research was also supported in part by a grant-in-aid for Scientific Research (S) from The Japan Society for Promotion of Sciences (Grant No. 17H06169) to Y. Asano. The financial support given to A. Nuylert from Ministry of Education, Culture, Sport, Science and Technology (MEXT) of Japan is deeply appreciated.

Author information

Author notes

  1. Zhenyu Zhai

    Present address: Center for Experimental Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, People’s Republic of China

  2. Zhenyu Zhai and Aem Nuylert have contributed equally to this work.

Authors and Affiliations

  1. Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

    Zhenyu Zhai, Aem Nuylert, Kimiyasu Isobe & Yasuhisa Asano

  2. Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu, Toyama, 939-0398, Japan

    Zhenyu Zhai, Aem Nuylert, Kimiyasu Isobe & Yasuhisa Asano

Authors
  1. Zhenyu Zhai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aem Nuylert
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kimiyasu Isobe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yasuhisa Asano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yasuhisa Asano.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 463 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhai, Z., Nuylert, A., Isobe, K. et al. Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris. J Ind Microbiol Biotechnol 46, 887–898 (2019). https://doi.org/10.1007/s10295-019-02162-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-019-02162-w

Keywords

Search

Navigation