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Development of a LAC4 promoter‐based gratuitous induction system in Kluyveromyces lactis
Author(s) -
Hsieh Huangpin Ben,
Da Silva Nancy A.
Publication year - 2000
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/(sici)1097-0290(20000220)67:4<408::aid-bit4>3.0.co;2-0
Subject(s) - inducer , galactokinase , galactose , lac operon , kluyveromyces lactis , beta galactosidase , plasmid , strain (injury) , chemistry , mutation , escherichia coli , biochemistry , gene , biology , microbiology and biotechnology , saccharomyces cerevisiae , anatomy
A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis . To prevent consumption of the inducer galactose, a strain with a gal1‐209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding β‐galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1‐209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high‐level induction. Under these conditions, β‐galactosidase specific and volumetric activities were 4.2‐ and 5.5‐fold higher, respectively, than those for the “ GAL1 ” nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter‐based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 408–416, 2000.