Hoshida Hisashi, Kidera Kenta, Takishita Ryuta, Fujioka Nobuhisa, Fukagawa Taiki, Akada Rinji
Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan; Research Center for Thermotolerant Microbial Resources, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan; Yamaguchi University Biomedical Engineering Center, 2-16-1 Tokiwadai, Ube 755-8611, Japan.
Department of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan.
J Biosci Bioeng. 2018 Jun;125(6):676-681. doi: 10.1016/j.jbiosc.2017.12.024. Epub 2018 Jan 20.
The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome. Inulinase is an enzyme hydrolyzing β-2,1-fructosyl bond in inulin and sucrose and is not required for xylose assimilation. Disruption of INU1 in the strain DMKU 3-1042 lost the production of the extracellular protein and resulted in growth defect in sucrose and inulin media, indicating that the extracellular protein was inulinase (sucrase). In addition, six K. marxianus strains among the 16 strains that were analyzed produced more inulinase in xylose medium than in glucose medium. However, expression analysis indicated that the INU1 promoter activity was lower in the xylose medium than in the glucose medium, suggesting that enhanced production of inulinase is controlled in a post-transcriptional manner. The production of inulinase was also higher in cultures with more agitation, suggesting that oxygen supply affects the production of inulinase. Taken together, these results suggest that both xylose and oxygen supply shift cellular metabolism to enhance the production of extracellular inulinase.
利用多种糖类的耐热酵母马克斯克鲁维酵母（Kluyveromyces marxianus）胞外蛋白的产生情况，通过使用含有葡萄糖、半乳糖和木糖等糖类的培养基进行了研究。对培养上清液的SDS-PAGE分析表明，当细胞在木糖培养基中生长时，会大量产生一种胞外蛋白。该胞外蛋白的N端序列与基因组中由INU1编码的菊粉酶的一部分相同。菊粉酶是一种水解菊粉和蔗糖中β-2,1-果糖基键的酶，木糖同化过程中不需要该酶。在菌株DMKU 3-1042中破坏INU1会导致胞外蛋白的产生丧失，并导致在蔗糖和菊粉培养基中生长缺陷，这表明该胞外蛋白是菊粉酶（蔗糖酶）。此外，在分析的16个菌株中，有6个马克斯克鲁维酵母菌株在木糖培养基中产生的菊粉酶比在葡萄糖培养基中更多。然而，表达分析表明，木糖培养基中INU1启动子活性低于葡萄糖培养基，这表明菊粉酶产量的增加是以转录后方式调控的。在搅拌更剧烈的培养物中菊粉酶的产量也更高，这表明氧气供应会影响菊粉酶的产生。综上所述，这些结果表明木糖和氧气供应都会改变细胞代谢，以提高胞外菊粉酶的产量。
