Send Orders for Reprints to reprints@benthamscience.ae 208 The Open Biotechnology Journal, 2016, 10, 208-222 1874-0707/16 2016 Bentham Open The Open Biotechnology Journal Content list available at: www.benthamopen.com/TOBIOTJ/ DOI: 10.2174/18740707016100100208 Characteristics of kanMX4-inserted Mutants that Exhibit 2- Deoxyglucose Resistance in Thermotolerant Yeast Kluyveromyces marxianus Suprayogi 1 , M. Nurcholis 1 , M. Murata 1 , N. Lertwattanasakul 2 , T. Kosaka 4 , N. Rodrussamee 1,3 , S. Limtong 2 and M. Yamada 1,4,* 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 Received: October 17, 2015 Revised: December 31, 2015 Accepted: February 3, 2016 Abstract: 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. Keywords: Glucose repression, 2-deoxyglucose-resistant mutants, Kluyveromyces marxianus, kanMX4 insertion, xylose fermentation. INTRODUCTION Ethanol as a bioethanol derived from lignocellulose is an environment-friendly alternative to fossil fuels. Since a substantial fraction of lignocellulose material consists of xylose and glucose [1], efficient bioconversion of xylose to ethanol is expected to make the process cost-effective [2]. Kluyveromyces marxianus is expected to become one of the best choices for industrial ethanol production for the following reasons. First, most K. marxianus strains are thermotolerant, and they are therefore capable of growing and fermenting at high temperatures, of which the optimal temperature is 10-15°C higher than that of the traditional alcohol- fermenting yeast Saccharomyces cerevisiae [3 - 5]. This thermal character has received much attention for the possibility of cost saving in ethanol production [ 6, 7]. Second, K. marxianus assimilates various sugars including glucose, xylose, arabinose, galactose, sucrose and inulin [ 7, 8]. Third, its growth is relatively rapid with a typical doubling time of 1.35-2 h [9, 10]. Fourth, the yeast has a high capacity of protein production [11]. K. marxianus is thus thought to have great promise for industrial applications and has been widely tested in various experiments for biotechnological applications, including ethanol production from whey or lactose [12 - 14], production of biomass or * Address correspondence to this author at the Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan; E-mail: m-yamada@yamaguchi-u.ac.jp