Hou Shengbo, Feng Hualiang, Gao Jiaoqi, Li Yimin, Yuan Wenjie, Bai Fengwu
School of Life Sciences and Biotechnology, Dalian University of Technology, Dalian 116023, Liaoning, China.
State Key Laboratory of Microbial Metabolism, Shanghai Jiaotong University, Shanghai 200240, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jun 25;33(6):923-935. doi: 10.13345/j.cjb.160458.
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，在生物燃料发酵中越来越受到关注。虽然这类酵母能够发酵戊糖，但发酵能力差异很大。比较了三株马克斯克鲁维酵母菌株（K. m 9009、K. m 1911和K. m 1727）在不同温度下对木糖和阿拉伯糖的发酵情况。结果表明，在不同发酵温度下，这三株菌株的发酵性能存在显著差异。特别是，K. m 9009和K. m 1727在40℃时的糖消耗率和酒精产量优于30℃。这一结果充分体现了马克斯克鲁维酵母在高温下的发酵优势。在此基础上，通过PCR扩增了三种不同酵母中编码关键代谢酶的五个基因（XR、XDH、XK、AR和LAD），并利用Clustalx 2.1对序列进行了比较。结果表明，编码关键酶的氨基酸序列与文献报道的参考序列相似度超过98%。此外，氨基酸差异并非在其酶的关键位点，因此三株菌株之间的差异不是由基因水平引起的，而是由转录或翻译调控水平引起的。通过实时PCR实验，我们测定了K. m 1727和K. m 1911在不同发酵时间点木糖代谢途径中四种关键酶（XR、XDH、XK和ADH）的基因表达水平。结果表明，对于耐热酵母K. m 1727，XDH和XK基因的低表达水平是导致木糖醇积累的主要因素。此外，根据NCBI和KEGG中已报道的拜耳接合酵母途径，确定了马克斯克鲁维酵母的木糖和阿拉伯糖代谢途径，为通过代谢工程进一步提高戊糖发酵能力奠定了基础。
