Abstract
Chymosin is an important industrial enzyme widely used in cheese manufacturing. Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, only low yields of chymosin (80 U mL(-1) in shake flask culture) have been obtained using K. lactis GG799. The aim of this study was to increase the amount of recombinant calf chymosin secreted by K. lactis GG799 by disrupting the PMR1 gene. Kluyveromyces lactis GG799 harbouring the disrupted PMR1 gene showed reduced growth in ethylene glycol tetraacetic acid-containing and Ca(2+) -deficient medium, but Ca(2+) supplementation eliminated the growth problem. The calf chymosin gene was ligated into the K. lactis GG799 expression vector, generating the plasmid pKLAC1-N-prochymosin. The linearised plasmid was homologously integrated into the genome of K. lactis GG799. In shake flask culture, chymosin activity was 496 U mL(-1) in the K. lactis PMR1-deficient mutant, sixfold higher than that in wild-type K. lactis GG799. Disrupting the PMR1 gene improved chymosin production in K. lactis GG799 sixfold. This knowledge could be applied to industrial chymosin production.
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