Skip to main content

Advertisement

SpringerLink
Log in

Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris

  • Applied Genetics and Molecular Biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

β-1,3-1,4-glucanase (EC3.2.1.73) as an important industrial enzyme has been widely used in the brewing and animal feed additive industry. To improve expression efficiency of recombinant β-1,3-1,4-glucanase from Bacillus licheniformis EGW039(CGMCC 0635) in methylotrophic yeast Pichia pastoris GS115, the DNA sequence encoding β-1,3-1,4-glucanase was designed and synthesized based on the codon bias of P. pastoris, the codons encoding 96 amino acids were optimized, in which a total of 102 nucleotides were changed, the G+C ratio was simultaneously increased from 43.6 to 45.5%. At shaking flask level, β-1,3-1,4-glucanase activity is 67.9 and 52.3 U ml−1 with barley β-glucan and lichenan as substrate, respectively. At laboratory fermentor level, the secreted protein concentration is approximately 250 mg l−1. The β-1,3-1,4-glucanase activity is 333.7 and 256.7 U ml−1 with barley β-glucan and lichenan as substrate, respectively; however, no activity of this enzyme on cellulose is observed. Compared to the nonoptimized control, expression level of the optimized β-1,3-1,4-glucanase based on preferred codons in P. pastoris shown a 10-fold higher level. The codon-optimized enzyme was approximately 53.8% of the total secreted protein. The optimal acidity and temperature of this recombinant enzyme were pH 6.0 and 45°C, respectively.

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
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Akio K, Takayuki B, Toshitaka M, Hirotsune M (2005) High expression of a synthetic gene encoding potato α-glucan phosphorylase in Aspergillus niger. J Biosci Bioeng 5:531–537

    Google Scholar 

  • Akira M, Yumiko TA (2001) Comparison of three signals for secretory expression of recombinant human midkine in Pichia pastioris. Biosci Biotechnol Biochem 65:2291–2293

    Article  Google Scholar 

  • Akita M, Kaytama K, Hatada Y, Ito S, Horikoshi K (2005) A novel β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol Lett 248:9–15

    Article  CAS  PubMed  Google Scholar 

  • Anjali Y, Christian FO (2003) Effect of codon optimization on expression levels of a functionally folded malaria vaccine candidate in prokaryotic and eukaryotic expression systems. Infect Immun:4961–4969 (Sept)

  • Ausubel FM, Brent R, Kingston RE, Moore DD (1999) Short protocols in molecular biology, 4th edn. Wiley, New York

    Google Scholar 

  • Bielecki S, Galas E (1991) Microbial β-glucanase different from cellulases. Crit Rev Biotechnol 10:275–305

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  CAS  PubMed  Google Scholar 

  • Chang SW, Lee GC, Shaw JF (2006) Codon optimization of Candadi rugosa lip1 for improving expression in Pichia patoris and biochemical characterization of the purified recombinant LIP1 lipase. J Agric Food Chem 3:815–822

    Article  CAS  Google Scholar 

  • Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as host system for transformation. Mol Cell Biol 5:3376–3385

    CAS  PubMed  PubMed Central  Google Scholar 

  • Delroisse JM, Marie D, Gilsoul JJ, Thami EM, Jacqueline D, Daniel P, Philippe T, Eric H, Micheline V (2005) Expression of a synthetic gene encoding a Tribolium castaneum carbosylesterase in Pichia pastoris. Protein Expr Purif 42:286–294

    Article  CAS  PubMed  Google Scholar 

  • Edward H, Wendy GB (1984) Expression of the cloned endo-1,3-1,4-glucanase gene of Bacillus subtilis in Saccharomyces cerevisiae. Curr Genet 8:471–475

    Article  Google Scholar 

  • Ekinci MS, Flint HJ (2001) Expression of bifunctional genes encoding xylanase and β(1,3-1,4)-glucanase in gram-positive bacteria. Turk J Vet Anim Sci 25:771–775

    Google Scholar 

  • Ekinci MS, Mccrae SI, Flint HJ (1997) Isolation and overexpression of a gene encoding an extracellular 1,3-1,4-glucanase from Streptococcus bovis. Appl Environ Microbiol 63:3752–3756

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fadi FH, Aisha M, Paula R (2002) Codon optimization improves heterologous expression of a Schistosoma mansoni cDNA in HEK293 cells. Parasitol Res 88:583–586

    Article  Google Scholar 

  • Hale RS, Thompson G (1998) Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli. Protein Expr Purif 12:185–188

    Article  CAS  PubMed  Google Scholar 

  • Higgins DR, Cregg JM (1998) Pichia protocols methods in molecular biology. Humana Press Inc, Totowa, NJ

    Book  Google Scholar 

  • Hovarth H, Huang J, Wong O, Kohl E, Okita T, Kannangara CG, von Wettstein D (2000) The production of recombinant proteins in transgenic barley grains. Proc Natl Acad Sci USA 97:1914–1919

    Article  Google Scholar 

  • Jaskari J, Kontula P, Sitonen A, Jousimies SH, Mattila ST, Poutanen K (1998) Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobaterium and Lactobacillus strains. Appl Microbiol Biotechnol 49:175–181

    Article  CAS  PubMed  Google Scholar 

  • Jensen LG, Olsen O, Kops O (1996) Transgenic barley expressing a protein- engineered, thermostable (1,3-1,4)-β-glucanase during germination. Proc Natl Acad Sci USA 93:3487–3491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jung HW, Yuan YL, Askale M, Scott S, Zhirui W, Jerry T, David MN (2002) Gene optimization is necessary to express a bivalent anti-human anti-T cell immunotoxin in Pichia pastoris. Protein Expr Purif 25:270–282

    Article  CAS  Google Scholar 

  • Kim JY (2003) Overproduction and secretion of Bacillus circulans endo-β-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium. Biotechnol Lett 25:1445–1449

    Article  CAS  PubMed  Google Scholar 

  • Li S, Sauer WC, Huang SX, Gabert VI (1996) Effect of beta-glucanase supplementation to hulless barley- or wheat-soybean meal diets on the digestibilities of energy, protein, beta-glucans and amino acid in young pigs. J Anim Sci 74:1649–1656

    Article  CAS  PubMed  Google Scholar 

  • Manuel M, Cecilia C, Lester H, Jose P, Laura V, Jorge V, Yanet T, Abdel H, Liuba P, Vladimir B, Liudys G, Emilio L, Lila C, Eduardo M (2005) Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnol Lett 27:339–345

    Article  CAS  Google Scholar 

  • Masaru K, Kazuhiro M, Makiko H, Takaichi H, Takeaki O, Sohkichi M, Takeshi Y, Shudo Y, Atsuhiko H, Naoki Y, Mitsuo H (2005) Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette–Guerin expressing human immunodeficiency virus type 1 gag. J Virol 8:8716–8723

    Google Scholar 

  • Mathlouthi N, Serge MS, Luc S, Bernard Q, Michel L (2002) Effects of xylanase and β-glucanase addition on performance, nutrient digestibility, and physico-chemical conditions in the small intestine contents and fecal microflora of broiler chickens fed a wheat and barley-based diet. Anim Res 51:395–406

    Article  CAS  Google Scholar 

  • Meldgaard M, Harthill J (1994) Different effect of N-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanase secreted from yeast. Microbiology 140:153–157

    Article  Google Scholar 

  • Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428

    Article  CAS  Google Scholar 

  • Nikolay SO, Willem JS, Maarten AJ (2002) Optimization of the expression of equistatin in Pichia pastoris. Protein Expr Purif 24:18–24

    Article  CAS  Google Scholar 

  • Oka C, Tanaka M, Muraki M, Harata K, Suzuki K, Jigami Y (1999) Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris. Biosci Biotechnol Biochem 11:1977–1983

    Article  Google Scholar 

  • Olsen O, Borriss R, Simon O, Thomsen KK (1991) Hybrid Bacillus (1-3,1-4)- β-glucanase: engineering thermostable enzymes by construction of hybrid genes. Mol Gen Genet 225:177–185

    Article  CAS  PubMed  Google Scholar 

  • Parrish FW, Perlin AS, Reese ET (1960) Selective enzymolysis of poly-β-D-glucan, and the structure of the polymers. Can J Chem 38:2094–2104

    Article  CAS  Google Scholar 

  • Piruzian ES, Monzavi KB, Darbinian NS (1998) The use of thermoactive β-glucanase gene from Clostridium thermocellum as a reporter gene in plants. Mol Gen Genet 257:561–567

    Article  CAS  PubMed  Google Scholar 

  • Planas A (2000) Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. BiochiM Biophys Acta 1543:361–382

    Article  CAS  PubMed  Google Scholar 

  • Prodromou C, Pearl HL (1992) Recursive PCR: a novel technique for total gene synthesis. Protein Eng 5:827–829

    Article  CAS  PubMed  Google Scholar 

  • Schimming S, Schwarz WH (1991) Properties of a thermoactive β-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli. Biochem Biophys Res Commun 17:447–452

    Article  Google Scholar 

  • Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190:55–62

    Article  CAS  PubMed  Google Scholar 

  • Sinclair G, Choy FYM (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  Google Scholar 

  • Stacey SP, Hebe MD, Rakesh KG, Gary SS (2005) Codon optimization of bacterial luciferase (lux) for expression in mammalian cells. J Ind Microbiol Biotechnol 32:115–123

    Article  CAS  Google Scholar 

  • Sue MP, Mariana LF, Brian M, Linda MH (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270

    Article  CAS  Google Scholar 

  • Teng D, Wang JH, Yao Y, Yang YL, Liu LH (2005) Cloning and expression of Bacillus licheniformis EG039 beta-1,3-1,4-glucanase in Pichia methanolica. In: Jianhua W (ed) Research and development of the new safe feed additives. China Science, Beijing, pp 102–109

    Google Scholar 

  • Teng D, Wang JH, Fan Y, Yang YL, Tian ZG, Luo J, Yang GP, Zhang F (2006) Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli. Appl Microbiol Biotechnol 72:705–712

    Article  CAS  PubMed  Google Scholar 

  • van Rensburg P, van Zyl WH, Pretorius IS (1997) Over-expression of the Saccharomyces cerevisiae exo-β-1,3-glucanase gene together with the Bacillus subtilis endo-β-1,3-1,4-glucanase gene and the Butyrivibrio fibrisolvens endo-β-1,4-glucanase gene in yeast. J Biotechnol 55:43–53

    Article  PubMed  Google Scholar 

  • Vervoort EB, Van Ravestein A, van Peij NN, Heikoop JC, van Hasstert PJ, Verheijden GF, Linskens MH (2000) Optimizing heterologous expression in Dictyostelium: importance of 5′ codon adaptation. Nucleic Acids Res 28:2069–2074

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Villatte F, Hussein AS, Bachmann TT (2001) Expression level of heterologous protein in Pichia pastoris is influenced by flask design. Appl Microbiol Biotechnol 55:463–465

    Article  CAS  PubMed  Google Scholar 

  • Wen TN, Chen JL, Lee SH, Yang NS, Shyur LF (2005) A truncated Fibrobacter succinogenes 1,3-1,4-β-glucanase with improved enzymatic activity and thermotolerance. Biochemistry 44:9197–9205

    Article  CAS  PubMed  Google Scholar 

  • Wolf M, Geczi A, Simon O, Borriss R (1995) Genes encoding xylan and β-glucan hydrolyzing enzymes in Bacillus subtilis: characterization, mapping and construction of strains deficient in lichenase, cellulase and xylanase. Microbiology 4:281–290

    Article  Google Scholar 

Download references

Acknowledgement

This study is supported by two Chinese National Hi-Tech R&D Programs (Chinese “863” Program No. 2001 AA 246041 and 2004 AA 246040). Da Teng and Ying Fan have contributed equally to this work.

Author information

Authors and Affiliations

  1. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China

    Da Teng, Ying Fan, Ya-lin Yang, Zi-gang Tian, Jin Luo & Jian-hua Wang

Authors
  1. Da Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ying Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ya-lin Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zi-gang Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jin Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jian-hua Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jian-hua Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Teng, D., Fan, Y., Yang, Yl. et al. Codon optimization of Bacillus licheniformis β-1,3-1,4-glucanase gene and its expression in Pichia pastoris . Appl Microbiol Biotechnol 74, 1074–1083 (2007). https://doi.org/10.1007/s00253-006-0765-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-006-0765-z

Keywords

Search

Navigation