Abstract
To analyze the glucose repression mechanism in the thermotolerant yeast Kluyveromyces marxianus, disrupted mutants of genes for Mig1 and Rag5 as orthologs of Mig1 and Hxk2, respectively, in Saccharomyces cerevisiae were constructed, and their characteristics were compared with those of the corresponding mutants of S. cerevisiae. MIG1 mutants of both yeasts exhibited more resistance than the corresponding parental strains to 2-deoxyglucose (2-DOG). Histidine was found to be essential for the growth of Kmmig1, but not that of Kmrag5, suggesting that MIG1 is required for histidine biosynthesis in K. marxianus. Moreover, Kmrag5 and Schxk2 were more resistant than the corresponding MIG1 mutant to 2-DOG, and only the latter increased the utilization speed of sucrose in the presence of glucose. Kmrag5 exhibited very low activities for gluco-hexokinase and hexokinase and, unlike Schxk2, showed very slow growth and a low level of ethanol production in a glucose medium. Furthermore, Kmrag5, but not Kmmig1, exhibited high inulinase activity in a glucose medium and exhibited greatly delayed utilization of accumulated fructose in the medium containing both glucose and sucrose. Transcription analysis revealed that the expression levels of INU1 for inulinase and GLK1 for glucokinase in Kmrag5 were higher than those in the parental strain; the expression level of INU1 in Kmmig1 was higher, but the expression levels of RAG1 for a low-affinity glucose transporter in Kmmig1 and Kmrag5 were lower. These findings suggest that except for regulation of histidine biosynthesis, Mig1 and Rag5 of K. marxianus play similar roles in the regulation of gene expression and share some functions with Mig1 and Hxk2, respectively, in S. cerevisiae.
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Acknowledgments
We thank K. Matsushita and T. Yakushi for their helpful discussion.
Funding
The Govermment of Indonesia gave financial support (to M. N) through BPPLN-DIKTI Scholarship, Ministry of Research, Technology, and Higher Education. This work was supported by The Core to Core Program A. Advanced Research Networks, which was granted by the Japan Society for the Promotion of Science, the National Research Council of Thailand, Ministry of Science and Technology in Vietnam, National Univ. of Laos, Univ. of Brawijaya, and Beuth Univ. of Applied Science Berlin and was also supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences, NEDO, Special coordination and by Japan Science and Technology Agency, Ministry of Research, Technology and Higher Education of the Republic of Indonesia, Agricultural Research Development Agency of Thailand and Ministry of Science and Technology of Laos as part of the e-ASIA Joint Research Program (e-ASIA JRP).
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MN obtained and carried out characterization of MIG1 and RAG5 disrupted mutants and performed enzyme assay, gene expression, and writing the manuscript. SN was involved in construction of MIG1 and RAG5 disrupted mutants. S was involved in obtaining mutant no. 23. SL isolated K. marxianus DMKU 3-1042. NR, NL, and TK were participated in discussion of the study. MY was contributed in the experimental design and discussion for writing the manuscript. All authors read and approved the final manuscript.
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Nurcholis, M., Nitiyon, S., Suprayogi et al. Functional analysis of Mig1 and Rag5 as expressional regulators in thermotolerant yeast Kluyveromyces marxianus. Appl Microbiol Biotechnol 103, 395–410 (2019). https://doi.org/10.1007/s00253-018-9462-y
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DOI: https://doi.org/10.1007/s00253-018-9462-y