Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.
马克斯克鲁维酵母作为非常规酵母在燃料乙醇发酵中受到人们越来越多的关注。马克斯克鲁维具有天然的发酵戊糖的能力，但不同菌株的发酵能力存在较大差异。本研究比较了3 株马克斯克鲁维菌株Kluyveromyces marxianus 9009/1911/1727 (K. m 9009/1911/1727) 在不同温度下的木糖和阿拉伯糖的发酵性能差异，结果发现不同发酵温度下，3 株菌在耗糖速率、糖醇产率均表现出了显著的差异。菌株K. m 9009 和K. m 1727在40 ℃下的发酵性能均优于30 ℃，这充分体现了马克斯克鲁维酵母的高温发酵优势。针对发酵差异，采用PCR 方法获得3 个不同菌株的戊糖代谢途径中的5 种关键代谢酶 (XR、XDH、XK、AR 和LAD) 的基因序列，并利用Clustalx 2.1 进行了序列比对。结果显示3 株菌的相关基因与文献中报道的1 株克鲁维酵母的相应关键酶氨基酸编码序列相似性达98%以上，并且差异的氨基酸不在酶的关键位点处。在此基础上，通过Real-time实验，对木糖发酵差异最为明显的K. m 1727 和K. m 1911 的木糖代谢过程4 个关键酶 (XR、XDH、XK 和ADH)的基因表达量进行测定，其结果显示对于耐热菌株K. m 1727，XDH 和XK 基因表达量低是导致木糖代谢过程中木糖醇积累、乙醇产量低的主要原因。最后，将所测得的马克斯克鲁维酵母的戊糖代谢关键酶序列与其他不同种属相比对，确定了其木糖和阿拉伯糖代谢途径，为进一步利用代谢工程方法提高戊糖发酵性能奠定了基础。.
Keywords: Kluyveromyces marxianus; arabinose fermentation; metabolic pathway; sequence aligment; xylose fermentation.