EFFECTS OF CULTIVATION CONDITIONS ON THE PRODUCTION OF HETEROLOGOUS ALPHA-GALACTOSIDASE BY KLUYVEROMYCES-LACTIS

Growth conditions relevant for the large-scale production of heterolog ous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassett e encoding guar alpha-galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possi ble to produce active extracellular alpha-galactosidase in shake-flask cultures grown on a defined mineral medium. inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. Wi th urea-containing mineral medium, enzyme production in shake flasks w as comparable to that in complex media containing peptone. In contrast , the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growin g on mineral media was not due to loss of the expression cassette, sin ce addition of peptone to such cultures resulted in an immediate high rate of alpha-galactosidase production. The discrepancy between the be haviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the sca ling-up of heterologous protein production from laboratory to producti on scale.