Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

✍ Scribed by Huangpin Ben Hsieh; Nancy A. Da Silva

• Book ID: 101243129
• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 150 KB
• Volume: 67
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(20000220)67:4<408::aid-bit4>3.0.co;2-0

✦ Synopsis

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 ␤-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, ␤-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.