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Enhanced production of leech hyaluronidase by optimizing secretion and cultivation in Pichia pastoris

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Abstract

Leech hyaluronidase (LHAase) was recently cloned and successfully expressed in Pichia pastoris. To increase its secretory expression level, four signal peptides (nsB, YTP1, SCS3, and HKR1) and six amphipathic peptides (APs) were comparatively investigated. After substitution with nsB and fusion with AP2, the production of LHAase was significantly increased, from 8.42 × 105 to 1.24 × 106 U/ml. Compared with the parental LHAase, the variant AP2-LHAase showed a lower optimum pH (5.0), higher optimum temperature (50 °C), and a broader range of thermal stability (20–60 °C). To further promote fermentative production of the variant AP2-LHAase, the cultivation temperature was systematically optimized according to cell viability and alcohol oxidase activity. Eventually, through a combination of N-terminal engineering and optimization of cultivation, the production of LHAase was improved to 1.68 × 106 U/ml, with a high productivity of 1.87 × 104 U/ml/h.

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References

  • Abad S, Kitz K, Hormann A, Schreiner U, Hartner FS, Glieder A (2010) Real-time PCR-based determination of gene copy numbers in Pichia pastoris. Biotechnol J 5:413–420

    Article  PubMed  CAS  Google Scholar 

  • Boltje TJ, Buskas T, Boons GJ (2009) Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research. Nat Chem 1:611–622

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • 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  PubMed  CAS  Google Scholar 

  • Celik E, Calik P (2012) Production of recombinant proteins by yeast cells. Biotechnol Adv 30:1108–1118

    Article  PubMed  CAS  Google Scholar 

  • Cereghino GP, Cereghino JL, Ilgen C, Cregg JM (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13:329–332

    Article  PubMed  Google Scholar 

  • Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52

    Article  PubMed  CAS  Google Scholar 

  • Damasceno LM, Huang CJ, Batt CA (2012) Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol 93:31–39

    Article  PubMed  CAS  Google Scholar 

  • El-Safory NS, Fazary AE, Lee CK (2010) Hyaluronidases, a group of glycosidases: current and future perspectives. Carbohydr Polym 81:165–181

    Article  CAS  Google Scholar 

  • Eldin P, Pauza ME, Hieda Y, Lin GF, Murtaugh MP, Pentel PR, Pennell CA (1997) High-level secretion of two antibody single chain Fv fragments by Pichia pastoris. J Immunol Methods 201(1):67–75

    Article  PubMed  CAS  Google Scholar 

  • Ferguson EL, Roberts JL, Moseley R, Griffiths PC, Thomas DW (2011) Evaluation of the physical and biological properties of hyaluronan and hyaluronan fragments. Int J Pharm 420:84–92

    Article  PubMed  CAS  Google Scholar 

  • Gao MJ, Zhan XB, Gao P, Zhang X, Dong SJ, Li Z, Shi ZP, Lin CC (2015) Improving performance and operational stability of porcine interferon-α production by Pichia pastoris with combinational induction strategy of low temperature and methanol/sorbitol co-feeding. Appl Biochem Biotechnol 176:493–504

    Article  PubMed  CAS  Google Scholar 

  • Gao Y, Zhao F, Wang Q, Zhang Y, Xu B (2010) Small peptide nanofibers as the matrices of molecular hydrogels for mimicking enzymes and enhancing the activity of enzymes. Chem Soc Rev 39:3425–3433

    Article  PubMed  CAS  Google Scholar 

  • Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M, Mattanovich D (2013) Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol 8:191–208

    Article  PubMed  CAS  Google Scholar 

  • Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750

    Article  PubMed  CAS  Google Scholar 

  • Ghose T (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268

    CAS  Google Scholar 

  • Guo X, Shi Y, Sheng J, Wang F (2014) A novel hyaluronidase produced by Bacillus sp. A50. PLoS One 9:e94156

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gurramkonda C, Polez S, Skoko N, Adnan A, Gäbel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Factories 9:31

    Article  CAS  Google Scholar 

  • Hasslacher M, Schall M, Hayn M, Bona R, Rumbold K, Lückl J, Griengl H, Kohlwein SD, Schwab H (1997) High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expr Purif 11:61–71

    Article  PubMed  CAS  Google Scholar 

  • Hofinger ES, Spickenreither M, Oschmann J, Bernhardt G, Rudolph R, Buschauer A (2007) Recombinant human hyaluronidase Hyal-1: insect cells versus Escherichia coli as expression system and identification of low molecular weight inhibitors. Glycobiology 17:444–453

    Article  PubMed  CAS  Google Scholar 

  • Holubova L, Korecka L, Podzimek S, Moravcova V, Rotkova J, Ehlova T, Velebny V, Bilkova Z (2014) Enhanced multiparametric hyaluronan degradation for production of molar-mass-defined fragments. Carbohydr Polym 112:271–276

    Article  PubMed  CAS  Google Scholar 

  • Hong F, Meinander NQ, Jönsson LJ (2002) Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnol Bioeng 79:438–449

    Article  PubMed  CAS  Google Scholar 

  • Jahic M, Wallberg F, Bollok M, Garcia P, Enfors SO (2003) Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microb Cell Factories 2:6

    Article  Google Scholar 

  • Jin P, Kang Z, Zhang N, Du G, Chen J (2014) High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers. Sci Rep 4:4471

    PubMed  PubMed Central  Google Scholar 

  • Kang Z, Yang S, Du G, Chen J (2014) Molecular engineering of secretory machinery components for high-level secretion of proteins in Bacillus species. J Ind Microbiol Biotechnol 41:1599–1607

    Article  PubMed  CAS  Google Scholar 

  • Kang Z, Zhang J, Jin P, Yang S (2015) Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes. Bioengineered 6:136–140

    Article  PubMed  Google Scholar 

  • Kozlov DG, Yagudin TA (2008) Antibody fragments may be incorrectly processed in the yeast Pichia pastoris. Biotechnol Lett 30:1661–1663

    Article  PubMed  CAS  Google Scholar 

  • Liang S, Li C, Ye Y, Lin Y (2013) Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris. Biotechnol Lett 35:97–105

    Article  PubMed  CAS  Google Scholar 

  • Ling Z, Ma T, Li J, Du G, Kang Z, Chen J (2012) Functional expression of trypsin from Streptomyces griseus by Pichia pastoris. J Ind Microbiol Biotechnol 39:1651–1662

    Article  PubMed  CAS  Google Scholar 

  • Ling Z, Liu Y, Teng S, Kang Z, Zhang J, Chen J, Du G (2013) Rational design of a novel propeptide for improving active production of Streptomyces griseus trypsin in Pichia pastoris. Appl Environ Microbiol 79:3851–3855

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Linker A, Hoffman P, Meyer K (1957) The hyaluronidase of the leech: an endoglucuronidase. Nature 180:810–811

    Article  PubMed  CAS  Google Scholar 

  • Lu X, Liu S, Zhang D, Zhou X, Wang M, Liu Y, Wu J, Du G, Chen J (2013) Enhanced thermal stability and specific activity of Pseudomonas aeruginosa lipoxygenase by fusing with self-assembling amphipathic peptides. Appl Microbiol Biotechnol 97:9419–9427

    Article  PubMed  CAS  Google Scholar 

  • Lünsdorf H, Gurramkonda C, Adnan A, Khanna N, Rinas U (2011) Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the hepatitis B surface antigen. Microb Cell Factories 10:48

    Article  CAS  Google Scholar 

  • Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270

    Article  PubMed  CAS  Google Scholar 

  • Macouzet M, Simpson BK, Lee BH (2005) Expression of a cold-adapted fish trypsin in Pichia pastoris. FEMS Yeast Res 5:851–857

    Article  PubMed  CAS  Google Scholar 

  • Reitinger S, Boroviak T, Laschober GT, Fehrer C, Müllegger J, Lindner H, Lepperdinger G (2008) High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris. Protein Expr Purif 57:226–233

    Article  PubMed  CAS  Google Scholar 

  • Sahara T, Gouda T, Tochigi Y, Ohgiya S (2007) A highly efficient secretory signal peptide and a protein expression system using the peptide thereof. Eur Pat: EP1790661

  • Sinha J, Plantz BA, Inan M, Meagher MM (2005) Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-tau. Biotechnol Bioeng 89:102–112

  • Stern R, Asari AA, Sugahara KN (2006) Hyaluronan fragments: an information-rich system. Eur J Cell Biol 85:699–715

    Article  PubMed  CAS  Google Scholar 

  • Stern R, Jedrzejas MJ (2006) Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev 106:818–839

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Stern R, Kogan G, Jedrzejas MJ, Soltes L (2007) The many ways to cleave hyaluronan. Biotechnol Adv 25:537–557

    Article  PubMed  CAS  Google Scholar 

  • Suye S, Ogawa A, Yokoyama S, Obayashi A (1990) Screening and identification of Candida methanosorbosa as alcohol oxidase-producing methanol using yeast. Agri Biol Chem 54:1297–1298

    Article  CAS  Google Scholar 

  • Vadhana AK, Samuel P, Berin RM, Krishna J, Kamatchi K, Meenakshisundaram S (2013) Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris. Enzym Microb Technol 52:177–183

    Article  CAS  Google Scholar 

  • Vanz AL, Lünsdorf H, Adnan A, Nimtz M, Gurramkonda C, Khanna N, Rinas U (2012) Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes. Microb Cell Factories 11:103

    Article  CAS  Google Scholar 

  • Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnol 30:385–404

    Article  CAS  Google Scholar 

  • Wang Q, Yang Z, Gao Y, Ge W, Wang L, Xu B (2008) Enzymatic hydrogelation to immobilize an enzyme for high activity and stability. Soft Matter 4:550

    Article  CAS  Google Scholar 

  • Wang Y, Wang Z, Xu Q, Du G, Hua Z, Liu L, Li J, Chen J (2009) Lowering induction temperature for enhanced production of polygalacturonate lyase in recombinant Pichia pastoris. Process Biochem 44:949–954

    Article  CAS  Google Scholar 

  • Werten MW, van den Bosch TJ, Wind RD, Mooibroek H, de Wolf FA (1999) High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15:1087–1096

    Article  PubMed  CAS  Google Scholar 

  • Woo JH, Liu YY, Stavrou S, Neville DM (2004) Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris. Appl Environ Microbiol 70:3370–3376

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yang H, Lu X, Liu L, Li J, Shin HD, Chen RR, Du G, Chen J (2013) Fusion of an oligopeptide to the N terminus of an alkaline α-amylase from Alkalimonas amylolytica simultaneously improves the enzyme's catalytic efficiency, thermal stability, and resistance to oxidation. Appl Environ Microbiol 79:3049–3058

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yuan P, Lv M, Jin P, Wang M, Du G, Chen J, Kang Z (2015) Enzymatic production of specifically distributed hyaluronanoligosaccharides. Carbohydr Polym 129:194–200

    Article  PubMed  CAS  Google Scholar 

  • Zhang J, Kang Z, Ling Z, Cao W, Liu L, Wang M, Du G, Chen J (2013) High-level extracellular production of alkaline polygalacturonate lyase in Bacillus subtilis with optimized regulatory elements. Bioresour Technol 146:543–548

    Article  PubMed  CAS  Google Scholar 

  • Zhang S, Holmes T, Lockshin C, Rich A (1993) Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci U S A 90:3334–3338

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zhou B, Xing L, Wu W, Zhang XE, Lin Z (2012) Small surfactant-like peptides can drive soluble proteins into active aggregates. Microb Cell Factories 11:10

    Article  CAS  Google Scholar 

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Acknowledgments

This work was financially supported by a grant from the Key Technologies R & D Program of Jiangsu Province, China (BE2014607); National High Technology Research and Development Program of China (863 Program, 2012AA022005); Program for Changjiang Scholars and Innovative Research Team in University (no. IRT1135); the National Science Foundation for Post-doctoral Scientists of China (2013M540414); the Jiangsu Planned Projects for Postdoctoral Research Funds (1301010B); and the 111 Project.

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Authors and Affiliations

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

    Zhen Kang, Na Zhang & Yunfeng Zhang

  2. Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China

    Zhen Kang

  3. School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Zhen Kang, Na Zhang & Yunfeng Zhang

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  1. Zhen Kang
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  2. Na Zhang
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Correspondence to Zhen Kang.

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Kang, Z., Zhang, N. & Zhang, Y. Enhanced production of leech hyaluronidase by optimizing secretion and cultivation in Pichia pastoris . Appl Microbiol Biotechnol 100, 707–717 (2016). https://doi.org/10.1007/s00253-015-7056-5

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