ENHANCEMENT OF RICE ALPHA-AMYLASE PRODUCTION IN RECOMBINANT YARROWIA-LIPOLYTICA

The productivity of recombinant rice alpha-amylase, a glycosylated pro tein with a molecular weight of 45 kDa, was improved by using a recomb inant Yarrowia lipolytica strain harboring an integrative vector, pXOS 103-In. While proteose peptone was found to be a good induer for the e xpression of alpha-amylase, ammonium sulfate was not. The optimal pH a nd specific growth rate for alpha-amylase production were 6.8 and 0.1 h(-1), respectively. The concentration of proteose peptone affected al pha-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 repre ssion, but alpha-amylase production resumed after the residual ammoniu m ion was depleted. When recombinant Y. lipolytica was grown in a cont rolled fed-batch culture in 1.5-l fermentor, using the bioprocess cont rol strategy developed in this work, a 28-fold increase in alpha-amyla se productivity was obtained (350 mg/l) as compared with that in a bat ch culture (12 mg/l). Practically all the alpha-amylase produced was s ecreted into the medium; only a negligible amount of intracellular alp ha-amylase was detected. Our bioprocess control strategy developed for a fed-batch culture using a recombinant Y. lipolytica strain can be a pplied to the overproduction of other recombinant proteins.