Document Type : Original Paper
Authors
1 1- Molecular Medicine Group, Medical Biotechnology Deapartement, National Institute of Genetics and Biotechnology, Tehran, Iran 2- Faculty of Basic Sciences, Islamic Azad University, Science and Research Branch, Tehran, Iran
2 ¹ Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Islamic Republic of Iran ³ Department of Bioprocess Engineering, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology
3 ³ Department of Bioprocess Engineering, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology
4 ³ Department of Bioprocess Engineering, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology² Faculty of Basic Sciences, Islamic Azad University, Science and Research Branch, Tehran, Islamic Republic of Iran
Abstract
Glucose oxidase (e.C1.1.3.4) is a flavoenzyme that catalyzes the oxidation of β-D-glucose to D-gluconolactone and hydrogen peroxide. The D-gluconolactone hydrolyzed spontaneously produces gluconic acid. Glucose oxidase has been widely used in chemical, food industries, and medicine. In this study, the glucose oxidase gene was identified in the chromosomal DNA of Aspergillus niger and isolated by PCR techniques. The glucose oxidase gene was then synthetically designed, optimized, cloned, and transferred into suitable hosts. The constructed clones were selected to modify the best production and combined promoter systems to produce glucose oxidase enzymes. In this design, the PCR product fragment was cloned into pPIC9 plasmids and transferred into Pichia pastoris GS115 by electroporation. The constructed strain was named pp.pPIC9/GOX. This strain was selected as a host for the other synthetic construct called pGAPZ/GOX. The strain pp.pPIC9/GOX was transformed as the host for the pGAPZ/GOX construct containing the glucose oxidase gene, which is controlled by the pGAPZa promoter with a self-inducing promoter and the newly constructed strain named pp.pPGAPZ/PIC9/GOX. The expression and comparison of the glucose oxidase activity were investigated by combining methanol and self-induction promoter in both pp.pPIC9/GOX and pp.pPGAPZ/PIC9/GOX strains simultaneously, which led to higher glucose oxidase gene activity in the new strain. Using the methanol induction promoter with the simultaneous expression of self-induction promoter in separate constructs and same strains increased the glucose oxidase gene activity.
Keywords
Main Subjects
- Kornecki JF, Carballares D, Tardioli PW, Rodrigues R, Murcia AB, Alcántara A, et al. Enzyme production of D-gluconic acid and glucose oxidase: successful tales of cascade reactions. Sci. Technol. 2020; 10: 5740-5771.
- Pazur JH, Kleppe K. the oxidation of glocuse and related compounds by glocuse oxidase from Aspergillus niger. Biochem.1964; 3: 578-583.
- Production of glucose oxidase in recombinant systems. 1992; US patent: 5094951.
- Rezaeepour N. Investigation of Cellulose Binding Heptapeptide Role in Immobilizing of Glucose Oxidase to the Nitro Cellulose surface [dissertation]. [Rasht]: University of Guilan, Department of Biology: Genetics; March 2019. 109p.
- Cherry JR, Berka RM, Halkier T. Recombinant expression of a glucose oxidase from a Cladosporium strain. 1999: US patent:
- Gomes AMV, Carmo TS, Carvalho LS, Bahia FM, Parachin Comparison of Yeasts as Hosts for Recombinant Protein Production. Microorganisms. 2018; 6(2): 38.
- Whittington H, Kerry-Williams S, Bidgood K, Dodsworth N, Peberdy J, Dobson M, Hinchliffe E, Balance D J. Expression of the Aspergillus niger glucose oxidase gene in A. niger, A. nidulance and Saccharomyces cerevisiae. Curr. Genet. 1990; 18:531-536.
- Kim MY, Chung HG, Hong SY, Kim HR, Lee JC, Park SM, Lee JH, Yang MS, Kim DH. Characterization of a novel allele of glucose oxidase from a Korean wild type strain of Aspergillus niger. Mol. cells. 2001; 11(3):2891-286.
- Bankar SS, Mahesh VB, Rekha SS, Laxmi. "Glucose oxidase—an overview". Biotechnol. Adv. 2009; 27(4):489-501.
- Anbu P, Subash CB, Gopinath A, Coleri C, Bidur P. "Microbial enzymes and their applications in industries and medicine". BioMed Res. Int. 2013; 2.
- Flynn N, Ranian A, Ramsey JD. Intracellular Delivery of Glucose Oxidase for Enhanced Cytotoxicity toward PSMA-Expressing Prostate Cancer Cells. Macromol Biosci. 2019 ;( 19)10: 1900183.
- Cereghinoa C, Geoff PL, Cereghinoa J, Lin I, James M. "Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris". Curr. Opin.Biotechnol. 2002; 13(4):329-332.
- Shen W, Kong C, Xue Y, Liu Y, Cai M, Zhan Y, et al. Kinase Screening in Pichia pastoris Identified Promising Targets Involved in Cell Growth and Alcohol Oxidase 1 Promoter (PAOX1) Regulation. Plos One. 2016; 11(12):
- Morrow Jr. "Improving protein production processes". Genet Eng. 2007; 27: 50-54.
- Heyland J, Fu J, Blank LM. "Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production". Bioeng. 2010; 107:357-368.
- Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, et al. "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. 2010; 9:9-49.
- Du C H, Xiao AJ. "Secretory expression and purification of the recombinant duck interleukin-2 in Pichia pastoris". J. Microbiol. Biotechnol. 2011; 21(12):1264-1269.
- Damasceno LM, Huang CJ, Batt CA. "Protein secretion in Pichia pastoris and advances in protein production". Appl. Microbiol. Biotechnol. 2012; 93:31-39.
- Soria T, Alvarez J, Manzano A, Relaño JL, Cases B, Mas-Fontao A et al. Mite allergoids coupled to nonoxidized mannan from Saccharomyces cerevisae efficiently target canine dendritic cells for novel allergy immunotherapy in veterinary medicine. Vet Immunol Immunopathol. 2017; 190: 65-72.
- Li P, Anumanthan A, Gao X G, Ilangovan K, Suzara VV, Düzgüneş N. "Expression of recombinant proteins in Pichia pastoris". Plasmid. 2007; 142(2):105-124.
- Daly R, Hearn MT. "Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production". J. Mol. Recognit. 2005; 18(2):119-138.
- Cregg J M, Vedvick T S, Raschke WC. "Recent advances in the expression of foreign genes in Pichia pastoris". Enzymol. 1993; 11(8):905-909.
- Houdebine LMJ. "Production of pharmaceutical proteins by transgenic animals". Comp Immunol Microbiol Infect Dis. 2009; 32:107-121.
- Shen W, Xue Y, Liu Y, Kong C, Wang X, Huanget M etal. A novel methanol-free Pichia pastoris system for recombinant protein expression. Microb Cell Fact. 2016; 15:178.
- Kunze G, Kang HA, Gellissen G. "Hansenula polymorpha (Pichia angusta): biology and applications", Yeast biotechnology: diversity and applications. Springer. 2009; 47-64.
- Cos P, Vlietinck AJ, Berghe DV, Maes LJ. "Anti-infective potential of natural products: how to develop a stronger in vitro ‘proof-of-concept’". Ethnopharmacol. 2006; 106(3), 290-302.
- Wang X, Sun Y, Ke F, Zhao H, Liu T, Xu L, Liu Y, Yan Ab. "Constitutive expression of Yarrowia lipolytica lipase LIP2 in Pichia pastoris using GAP as promoter". J. Biol. Chem. 2012; 263: 6209-6214.
- Ahmad M, Hirz M, Pichler H, Schwab Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol. 2014; 98(12): 5301–5317.
- Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY. "Recent advances on the GAP promoter derived expression system of Pichia pastoris". Mol. Biol. Rep. 2009; 36:1611-1619.
- Chen, CC, Wu PH, Huang CT, Change KJ. "A Pichia pastoris fermentation strategy for enhancing the heterologous expression of an Escherichia coli phytase". Enzyme Microb. Technol. 2004; 35(4):315-320.
- Wu JM, Hsu TA, Lee CK. "Expression of the gene coding for bacterial hemoglobin improves β-galactosidase production in a recombinant Pichia pastoris". Lett. 2003;25:1457-1462
- Macauley‐Patrick S, Fazenda ML, McNeil B, Harvey LM. "Heterologous protein production using the Pichia pastoris expression system". Yeast. 2005; 22:249-270.
- Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 2000; 24(1):45-66.
- Portela RMC, Vogl T, Ebner K, Oliveira R, Glieder A. Pichia pastoris Alcohol Oxidase 1 (AOX1) Core Promoter Engineering by High Resolution Systematic Mutagenesis. Biotechnol J. 2018;13(3):e1700340
- Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA. Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol. Cell. Biol. 1989;9(3): 1316-1323.
- Zhang W, Inan M, Meagher MM. Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris. Bioprocess Eng. 2000;5(4):275-287.
- Vogl T, Ruth C, Pitzer J, Kickenweiz, Glieder A. Synthetic Core Promoters for Pichia pastoris. ACS Synth. Biol. 2014; 3(3): 188–191
- Shen S, Sulter G, Jeffries TW, Cregg JM. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene.1998;216(1): 93-102.
- Kern A, Hartner FS, Freigassner M, Spielhofer J, Rumpf C, Leitner L, Glieder A. Pichia pastoris ‘just in time’alternative respiration. Microbiology. 2007; 153(4):1250-1260.
- Whitaker WB, Jones JA, Bennett RK, Gonzalez JE, Vernacchio VR, Collins SM et al. Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli. Metab. Eng. 2017; 39: 49-59.
- Gomes MZV, Palmqvist A. Immobilization of formaldehyde dehydrogenase in tailored siliceous mesostructured cellular foams and evaluation of its activity for conversion of formate to formaldehyde. Colloids Surf. B Colloid Surface B. 2018;163:41- 46.
- Gellissen G. Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 2000;54(6): 741-750.
- Egli T, Van Dijken J, Veenhuis M, Harder W, Fiechter A. Methanol metabolism in yeasts: regulation of the synthesis of catabolic enzymes. Arch. Microbiol. 1980;124(2-3):115-121.
|
|
- Sibirny A, Titorenko V, Efremov B, Tolstorukov I. Multiplicity of mechanisms of carbon catabolite repression involved in the synthesis of alcohol oxidase in the methylotrophic yeast Pichia pinus. Yeast. 1987;3(4):233-241.
- Krainer F W, Dietzsch C, Hajek T, Herwig C, Spadiut O, Glieder A. Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb. Cell Fact. 2012;11(1):1.
- Kriechbaum M, Heilmann HJ, Wientjes FJ, Hahn M, Jany KD, Gassen HG, Sharif F, Alaeddinglu G. Cloning and DNA sequence analysis of glucose oxidase gene from Aspergilles niger NRRL-3. FEBS Lett. 1989;255(1):63-66.
- Li-ping Z, Jing-hua Y, Tian-ran L, Yu-qi Y, He-ling Z. Late blight resistant transgenic potato expressing glucose oxidase gene. J. Agri. Univer. Hebei. 2001.
- Cassago A, Panepucci RA, Tortella Baiao AM, Henrique-Silva F. Cellophane based mini-prep method for DNA extraction from the filamentous fungus Trichoderma Reese, BMC Microbiol. 2002;2(14).
- Belyad F, Karkhanei AA, Raheb J. "Expression, characterization and one step purification of heterologous glucose oxidase gene from Aspergillus niger ATCC 9029 in Pichia pastoris" EuPA Open Proteom. 2018;19:1.
- Gu L, Zhang J, Liu B, Du G, Chen J. High-level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy. Appl. Biochem. Biotechnol. 2015; 175(3):1429-1447.
)