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Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed‐batch culture
Author(s) -
Panuwatsuk Wannana,
Da Silva Nancy A.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10518
Subject(s) - inducer , extracellular , kluyveromyces lactis , beta galactosidase , intracellular , fed batch culture , lactococcus lactis , galactose , biology , expression vector , biochemistry , escherichia coli , lac operon , microbiology and biotechnology , fermentation , chemistry , recombinant dna , yeast , bacteria , saccharomyces cerevisiae , gene , lactic acid , genetics
A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed‐batch culture. The Escherichia coli lacZ gene (β‐galactosidase; intracellular) and MFα1 leader‐ BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial‐pKD1 plasmids, and transformed into a ga1‐209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both β‐galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake‐flask culture, biomass concentration, β‐galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed‐batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5‐fold increase in biomass, a 23‐fold increase in β‐galactosidase activity, and a 3‐fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high‐cell‐density fed‐batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 712–718, 2003.