Expression Of The Aspergillus Niger Glucose Oxidase Gene In Pichia Pastoris SMD1168

Glucose oxidase (GOD) is a kind of aerobic-dehydrogenase. GOD catalyzes the β-D-glucose to D-gluconolactone and generates hydrogen peroxide with molecular oxygen as an electron acceptor. GOD has widely been applied in biomedicine, glucose quantitative analysis, medical testing, food industry, and biosensors. Natural GOD is mostly expressed in animals, plants and microganisms. While levels of GOD produced from the former two sources and from bacteria are very low. At present, GOD is mainly prepared and purificated from Aspergilus niger and Penicillium, but both the low productivity and the complicated purification process limit its applification. It has been attached great importance to construct better GOD production strain using genetic engineering technologies.Pichia pastoris expression system is one of the most common used eukaryotic expression systems for exogenous gene expression, which produces high levels of secretory heterologous proteins, and is simply to be purified. In addition to strong and strictly regulating the expression of the heterologous proteins, P. pastoris possesses advantages in regulating the expression proteins for processing, folding, post-translational modification, and secreting proteins to extracellular medium. Moreover, compared with other eukaryotic expression systems, P. pastoris expression system is more efficient, simple, and cost-effective.Promoters are cis-acting elements of regulation of gene expression and are also the key elements that influence gene expression. Promoters can significantly affect the level of heterologous proteins expressed in P. pastoris and are responsible for the space and time orders in gene expression. But the ability to express specific heterologous proteins in different P. pastoris strains are not the same. Therefore it is very important to choose suitable promoters to increase gene expression and facilitate the production of foreign proteins in P. pastoris. The plasmid pGAPZaA is a kind of secretory expression vector, carrying3-glyceraldehyde phosphate dehydrogenase gene promoter (pGAP), which can constitutively express exogenous proteins in P. pastoris without any inducer.In this study, the GOD gene from A. niger accc30161was cloned to construct the secretory expression vector with pGAP, named as pGAPZaA-GOD. In order to compare the different expression level between different P. pastoris strains under the control of pGAP, the vectors were transformed into P. pastoris GS115, KM71, and SMD1168strains respectively.Methods:1. Extraction of A. niger genomic DNA followed by amplification and sequencing of A. niger GOD gene;2. Construction, electrophoresis anaylysis, PCR, restriction digestion anaylysis and sequencing of recombinant plasmid between GOD gene and the secretory expression vector pGAPZaA, named as pGAPZaA-GOD;3. Transformation of the recombinant plasmid pGAPZaA-GOD into three different P. pastoris strains, GS115, KM71, and SMD1168. PCR was performed to identify the resulted transformants, GS115-GOD, KM71-GOD, and SMD1168-of GOD respectively;4. SDS-PAGE analysis of GOD;5. GOD enzyme activity assay;6. Influences of temperature and medium pH on GOD activity.Results:1. Analysis of sequencing and comparing showed that the obtained DNA fragment contained A. niger GOD gene, of which length1818bp without intron sequences and encoded605amino acids; 2. The P. pastoris expression vector pGAPZaA-GOD with A. niger accc30161GOD gene was successfully constructed;3. The transformant SMD1168-GOD was obtained;4. Result of SDS-PAGE analysis showed that the recombinant yeasts could express A. niger GOD protein.5. The transformant SMD1168-GOD could express active GOD;6. Temperature and medium pH could significantly influence the expression of GOD in P. pastoris expression systems.Conclusion:The expression vector pGAPZaA-GOD containing the Aspergillus niger GOD gene was successfully constructed. After electroporation, the pGAPZaA-GOD related DNA fragment was integrated into genome of Pichia pastoris SMD1168. The recombinant Pichia pastoris SMD1168-GOD could express active Aspergillus niger GOD at high level. The activity of GOD in the culture supernatant of SMD1168-GOD could be up to107.18u/mL at the condition of30℃and pH6.