Hsieh H B, Da Silva N A
Department of Chemical and Biochemical Engineering & Materials Science, University of California, Irvine, California 92697-2575, USA.
Biotechnol Bioeng. 2000 Feb 20;67(4):408-16. doi: 10.1002/(sici)1097-0290(20000220)67:4<408::aid-bit4>3.0.co;2-0.
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 beta-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, beta-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.
为乳酸克鲁维酵母开发了一种基于强大的内源LAC4启动子的免费诱导系统。为了防止诱导剂半乳糖的消耗，使用了具有gal1-209突变的菌株；这种突变使半乳糖激酶功能失活，但保留了诱导的调节功能。大肠杆菌lacZ基因（编码β-半乳糖苷酶）在乳酸克鲁维酵母中具有功能，并被用作多拷贝质粒上LAC4启动子下游的报告基因。gal1-209菌株表现出几种意想不到的现象，包括诱导剂半乳糖的部分消耗（尽管相对于GAL1菌株来说消耗速度要慢得多）以及在高浓度半乳糖下的生长抑制。然而，这些不寻常的特性并没有妨碍成功构建一个强大的免费诱导系统。由于免费菌株诱导剂的消耗率较低，极低浓度的半乳糖（半乳糖与葡萄糖的比例为1:20）就能导致高水平的诱导。在这些条件下，β-半乳糖苷酶的比活性和体积活性分别比“GAL1”非免费菌株高4.2倍和5.5倍。这项研究证明了通过基于LAC4启动子的免费诱导（从而获得更稳定的诱导剂浓度）可以提高生产力。还确定了各种碳源浓度对生长和诱导的影响。
