Expression of aspartic protease from Neurospora crassa in industrial ethanol-producing yeast and its application in ethanol production

In order to further improve the utilization rate of raw materials and increase ethanol productivity, the gene Asp, encoding aspartic protease in Neurospora crassa was cloned and expressed in industrial ethanol-producing yeast. To promote secretion of the acid protease, the gene was fused to signal sequence of the yeast α-factor gene and constitutively expressed under transcriptional control of the Saccharomyces cerevisiae PGK1 promoter. The resultant recombinant enzyme was characterized with respect to pH and temperature optimum. Then, this acid protease was anchored to the yeast cell wall by fusing the mature protein to the α-agglutinin peptides. The resultant strain was evaluated in clarifying corn mash and very high gravity (VHG) raw starch fermentation. The present results demonstrated that expression of the acid protease increased both growth rate and viable yeast counts and the recombinant strain of S. cerevisiae exhibited a higher ethanol yield as compared to the parent strain.