Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal ��-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.

Improving the Catalytic Performance of a Talaromyces leycettanus α-Amylase by Changing the Linker Length

“…Purified Amy13A, Amy13A21, and Amy13A33 displayed optimal activities at pH 4.5, while the pH optimum of Amy13A-CBM was measured at pH 5.0 (Figure 4A). Their pH optima fall within the pH range of fungal α-amylases (pH 4.0−7.0) and are similar to those of the α-amylases from Aspergillus oryzae (pH 5.0) 31 and Aspergillus awamori (pH 4.8). 32 Similar to the GH13 α-amylase from Penicillium expansum that has an optimal temperature of 60 3.…”

Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae

“…To improve the production of heterologous proteins, in addition to reducing the activity of proteases, a series of strong promoters were developed to induce the expression of heterologous genes (Table 2). In A. oryzae, the starch-inducible promoter of the alpha-amylase gene (PamyB) is one of the most commonly used promoters for the production of foreign proteins (Tsuchiya et al, 1992;Ward et al, 1992;Rey et al, 2003;Hoshida et al, 2005;Yamashita et al, 2011;Yin et al, 2015). The glucoamylase gene promoter (PglaA) and its improved promoter (PglaA142) were often used for the production and function analysis of recombinant proteins, such as Candida antarctica lipase B and A. oryzae recombinant tannase (Tamalampudi et al, 2007;Tokuoka et al, 2008;Ichikawa et al, 2020).…”