Characteristics of kanMX4-inserted Mutants that Exhibit 2-Deoxyglucose Resistance in Thermotolerant Yeast Kluyveromyces marxianus
Suprayogi1, M. Nurcholis1, M. Murata1, N. Lertwattanasakul2, T. Kosaka4, N. Rodrussamee1, 3, S. Limtong2, M. Yamada1, 4, *
1 Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Ube, 755-8505, Japan
2 Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
3 Faculty of Science, Chiangmai University, Chiangmai 50200, Thailand
4 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan
Kluyveromyces marxianus has the attractive potential of utilization capability of various sugars in addition to thermal tolerance and protein productivity. The yeast, however, has an intrinsic system for catabolite repression, by which cells down-regulate the metabolism of alternative sugars when glucose coexists. To acquire glucose-repression-free mutants, we isolated and characterized 2-deoxyglucose-resistant mutants from kanMX4-inserted mutants. The insertion site was determined by TAIL-PCR followed by nucleotide sequencing, indicating that the kanMX4 cassette was intragenically or intergenically inserted. Further analysis of the sugar utilization ability allowed to classify the intragenically inserted mutants including the mig1 mutant into two categories. One group showed enhanced utilization of xylose in the presense of glucose, presumably due to a defect in the glucose-repression mechanism, and the other group showed delayed utilization of glucose, probably by reduction of the uptake or initial catabolism of glucose. Considering the possible functions of the disrupted genes in these mutants, it is assumed that K. marxianus has undiscovered mechanisms for glucose repression and complex regulation for the uptake or initial catabolism of glucose.
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* Address correspondence to this author at the Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan; E-mail: firstname.lastname@example.org