title: Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture.
abstract: 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 (beta-galactosidase; intracellular) and MFalpha1 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 beta-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, beta-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 beta-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.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of California at Irvine, Irvine, California 92697-2575, USA.
authors.forename: Wannana
authors.initials: W
authors.lastname: Panuwatsuk
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Da Silva
mesh_terms: D007611:Aprotinin / Q000096:biosynthesis* / Q000235:genetics; D019149:Bioreactors; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005690:Galactose / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D016297:Mutagenesis, Site-Directed; D010865:Pilot Projects; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D007611:Aprotinin; D001616:beta-Galactosidase; D005947:Glucose; D005690:Galactose