title: Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal α-Amylase Gene in Aspergillus oryzae.
abstract: The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30°C. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.
authors: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Yanchen
authors: Y
authors: Yin
authors: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Youzhi
authors: Y
authors: Mao
authors: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Xiaolie
authors: X
authors: Yin
authors: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Bei
authors: B
authors: Gao
authors: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P.R. China.
authors: Dongzhi
authors: D
authors: Wei
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics; D001236:Aspergillus oryzae / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D017930:Genes, Reporter; D020459:Genes, rRNA; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010407:Penicillium / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D012331:RNA, Fungal / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D013696:Temperature; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
chemical_list: D004271:DNA, Fungal; D004275:DNA, Ribosomal; D012331:RNA, Fungal; D012335:RNA, Ribosomal; C065096:RNA, ribosomal, 26S; D011994:Recombinant Proteins; D000516:alpha-Amylases
keywords: Aspergillus oryzae; Penicillium sp.; heterologous expression; α-amylase