Functional Identification & Application In Strain Improvement Of Aspergillus Niger P_glaA

Glucoamylase is one of the most important industrial enzymes. It has been widely applied in fermentation industry that need glycosylated starch as a raw material. Aspergillus niger F0410 is an industrial glucoamylase producing strain. This research focused on cloning, sequence analyzing and functional identification of glaA promoter from A. niger F0410. The application of this promoter in improving the glucoamylase yield of A. niger F0410 was further carried out and discussed.Fristly, the PglaA fragment was amplified from the chromosomal DNA of A. niger F0410 by polymerase chain reaction. The DNA sequence was further sequenced. Sequence alignment with the PglaA sequence of another A. niger (GenBank accession number X00712) showed that the homologous of two promoter nucleotide sequences reached 99.45 percent, five sites of PglaA sequence has been changed, including one-site loss and four sites replacement.The PglaA was subsequently cloned into the up-stream of the KmR gene encoding region of the plasmid pRS303K to replace the promoter of KmR, yielded the hybrid plasmid pRS-PglaA-KmR. The recombinant plasmid was transformed into Escherichia coli JM109 cells, resulted in the recombinant strain E. coli(pRS-PglaA-KmR). That the activity of KmR was detectable in LB plate containing kanamycin showed that PglaA has function in E. coli. Glucose, sucrose, lactose, maltose or corn starch could enhance the strength of PglaA with defferent degrees.For strain improvement, the recombinant plasmid pRS-PglaA-HygR was constructed and genetic transformed into A. niger F0410 using a PEG-mediated method. With a selectable marker gene encoding hygromycin resistance, a cluster of transformants with pRS-PglaA-HygR randomly integrated into the genome of A. niger F0410 were gained and confirmed by diagnosis PCR.A transformant with superior character, named GAH14, is selected. After NTG mutagenizing, the mutant strains with improved hygromycin resistance are picked out for further fermentation study. After screening for several times, we obtain a strain named GAN12, and the activity of its glucoamylase 8.8% greater than the activity of initial strain.