Enhancement of rice α-amylase production in recombinant Yarrowia lipolytica

Abstract

The productivity of recombinant rice α-amylase, a glycosylated protein with a molecular weight of 45 kDa, was improved by using a recombinant Yarrowia lipolytica strain harboring an integrative vector, pXOS103-In. While proteose peptone was found to be a good induer for the expression of α-amylase, ammonium sulfate was not. The optimal pH and specific growth rate for α-amylase production were 6.8 and 0.1 h −1, respectively. The concentration of proteose peptone affected α-amylase expression when glycerol was used as a carbon source. The optimal ratio of glycerol to proteose peptone was found to be 2.5 for the fed-batch process. In a controlled fed-batch culture, addition of ammonium sulfate by pulse feeding in the production phase caused repression, but α-amylase production resumed after the residual ammonium ion was depleted. When recombinant Y. lipolytica was grown in a controlled fed-batch culture in 1.5- l fermentor, using the bioprocess control strategy developed in this work, a 28-fold increase in α-amylase productivity was obtained (350 mg/ l) as compared with that in a batch culture (12 mg/ l). Practically all the α-amylase produced was secreted into the medium; only a negligible amount of intracellular α-amylase was detected. Our bioprocess control strategy developed for a fed-batch culture using a recombinant Y. lipolytica strain can be applied to the overproduction of other recombinant proteins.