Heterologous phytase expression in the food filamentous fungus <i> Aspergillus oryzae </i>using the added rice husk cultivation model
Keywords:Aspergillus oryzae, phytase, DsRed, recombinant phytase, rice husk.
Aspergillus oryzae, a safe filamentous fungus, is widely used in food and enzyme production. In this study, we examined a cultivation model using rice husks as carrier to assess the capacity of recombinant protein production in A. oryzae. The model was first tested with the A. oryzae strain expressing the DsRed reporter gene. Expression of DsRed was easily detected by the pink color of the fungal mycelium on culture media and under a fluorescence microscope. The model was then evaluated with the phyA gene encoding a phytase from the fungus Aspergillus fumigatus. The phyA expression cassette regulated by the amyB promoter was permanently integrated into the genome of A. oryzae via Agrobacterium tumefaciens-mediated transformation with the pyrG nutritional marker. Results showed that A. oryzae transgenic strains carrying 2−3 copies of the phyA gene in their genomes exhibited a significant increase in phytase activity on agar medium supplemented with phytate. With rice husks added, these transgenic strains could secrete the recombinant phytase into the culture and phytase activity of the crude enzyme solution increased by 4.3 times compared to the unstransgenic A. oryzae. The established cultivation model and the transgenic approach in this study represent a potential for being used in production of secreted recombinant enzymes for animal feeds.
Barbesgaard P., Heldt-Hansen H. P., Diderichsen B., 1992. On the safety of Aspergillus oryzae: a review. Appl. Microbiol. Biotechnol., 36: 569−572.
Christensen T., Woeldike H., Boel E., Mortensen S.B., Hjortshoej K., Thim L., Hansen M.T., 1988. High level expression of recombinant genes in Aspergillus oryzae. Nat. Biotechnol., 6: 1419−1422.
Haefner S., Knietsch A., Scholten E., Braun J., Lohscheidt M., Zelder O., 2005. Biotechnological production and application of phytases. Appl. Microbiol. Biotechnol., 68: 588−597.
Heinonnen J. K., Lahti R. J., 1981. A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal. Biochem., 113: 313−317.
Huge-Jensen B., Andreasen F., Christensen T., Christensen M., Thim L., Boel E., 1989. Rhizornucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae. Lipids, 24: 781−785.
Li M., Zhou L., Liu M., Huang Y., Sun X., Lu F., 2013. Construction of an engineering strain producing high yields of α-transglucosidase via Agrobacterium tumefaciens - mediated transformation of Aspergillus niger. Biosci. Biotechnol. Biochem., 77: 1860−1866.
Machida M., Yamada O., Gomi K., 2008. Genomics of Aspergillus oryzae: learning from the history of koji mold and exploration of its future. DNA Res., 15: 173−183.
Michielse C. B., Hooykaas P. J. J., van den Hondel C. A. M. J. J., Ram A. F. J., 2005. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr. Genet., 48:1−17.
Nguyen T. K., Ho N. Q., Pham H. T., Phan T. N., Tran V. T., 2016. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae. World J. Microbiol. Biotechnol., 32: 204.
Nguyen T. K., Ho Q. N., Do L. T. B. X., Mai L. T. D., Pham D. N., Tran H. T. T., Le D. H., Nguyen H. Q., Tran V. T., 2017. A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation. World J. Microbiol. Biotechnol., 33: 107.
Pasamontes L., Haiker M., Wyss M., Tessier M., van Loon A. P. G. M., 1997. Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Appl. Environ. Microbiol., 63: 1696−1700.
Punt P. J., van Biezen N., Conesa A., Albers A., Mangnus J., van der Hondel C., 2002. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol., 20: 200−206.
Rao D. E. C. S., Rao K. V., Reddy T. P., Reddy V. D., 2009. Molecular characterization, physicochemical properties, known and potential applications of phytases: an overview. Crit. Rev. Biotechnol., 29: 182−198.
Schmittgen T. D., Livak K. J., 2008. Analyzing real-time PCR data by the comparative Ct method. Nat. Protoc., 3: 1101−108.
Tsuchiya K., Tada S., Gomi K., Kitamoto K., Kumagai C., Tamura G., 1992. Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae. Biosci. Biotechnol. Biochem., 56: 1849−1853.
Varga J., Frisvad J. C., Samson R., 2011. Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Stud. Mycol., 69: 57−80.
White T. J., Bruns T., Lee S. J. W. T., Taylor J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications, 18: 315−322.
Zhu L., Maruyama J. I., Kitamoto K., 2013. Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae. Appl. Biochem. Biotechnol., 97: 6347−6357.