Chen Yulei, Tang Jiaye, Li Qian, Xin Wenli, Xiao Ximeng, Mou Borui, Li Jialian, Lu Fujia, Fu Chun, Long Wencong, Liao Hong, Han Xuebing, Feng Peng, Li Wei, Zhou Kedi, Yang Liuyun, Chen Xuemei, Yang Lixi, Ma Menggen, Yang Yaojun, Wang Hanyu
Bamboo Diseases and Pests Control and Resources Development Key Laboratory of Sichuan ProvinceCollege of Life Science, Leshan Normal University, No. 778 Binhe Road, Leshan, 614000, Sichuan, China.
College of Resources, Sichuan Agricultural University, Wenjiang, Chengdu, 611130, Sichuan, China.
Arch Microbiol. 2025 Jun 26;207(8):188. doi: 10.1007/s00203-025-04378-0.
Levulinic acid (LA) is the main toxic by-product in the production of fuel ethanol, and its large-scale emission adversely affect the ecological environment. In order to effectively remove LA from the liquid waste, microbial degradation methods are adopted but the challenge is that microorganisms cannot fully tolerate LA in the waste. Therefore, it is particularly important to explore the tolerance mechanism of microorganisms to LA. In this study, the whole-genome knockout library scanning and sensitive knockout strain identification were carried out. In addition, subcellular structures such as mitochondria, vacuoles, and endoplasmic reticulum as well as reactive oxygen species (ROS) accumulation were observed under a fluorescence microscopy after stained with fluorescent dyes such as 2'7'-DCF diacetate, Mito Tracker Green FM, Vacuole Membrane Marker MDY-64, and ER-Tracker Red dye. We also performed genomic sequencing on the wild-type strain and knockout strain. Through comparative genomic analysis, it's been found that the LDH1 (YBR204C) gene in Saccharomyces cerevisiae helps promote the clearance of intracellular reactive oxygen species, and the deletion of LDH1 leads to a more-than-two-fold down-regulation of genes related to cell membrane, cell wall, and cell cycle. By measuring the transcriptome and metabolome of the LDH1 knockout strain (ldh1Δ) under LA stress and comparing it with the wild-type strain BY4741, we found that under the condition of LDH1 knockout, the accumulation of NAD intermediates would be activated, disrupting normal cell functions and causing cell damage. The LDH1 gene knockout also affects the Methyl Cycle in the cell, which is closely related to the accumulation of ROS. These research results make it possible to create a new genetically modified strain of S. cerevisiae with desired higher tolerance which enhances degradation efficiency and reduces cost.
乙酰丙酸（LA）是燃料乙醇生产中的主要有毒副产物，其大规模排放对生态环境产生不利影响。为了有效去除废液中的LA，采用了微生物降解方法，但挑战在于微生物不能完全耐受废液中的LA。因此，探索微生物对LA的耐受机制尤为重要。在本研究中，进行了全基因组敲除文库扫描和敏感敲除菌株鉴定。此外，在用2'7'-二氯荧光素二乙酸酯、线粒体追踪绿荧光染料、液泡膜标记物MDY-64和内质网追踪红染料等荧光染料染色后，在荧光显微镜下观察线粒体、液泡和内质网等亚细胞结构以及活性氧（ROS）的积累情况。我们还对野生型菌株和敲除菌株进行了基因组测序。通过比较基因组分析，发现酿酒酵母中的LDH1（YBR204C）基因有助于促进细胞内活性氧的清除，而LDH1的缺失导致与细胞膜、细胞壁和细胞周期相关的基因下调超过两倍。通过测量LA胁迫下LDH1敲除菌株（ldh1Δ）的转录组和代谢组，并将其与野生型菌株BY4741进行比较，我们发现，在LDH1敲除的情况下，NAD中间体的积累会被激活，扰乱正常细胞功能并导致细胞损伤。LDH1基因敲除还影响细胞中的甲基循环，这与ROS的积累密切相关。这些研究结果使得有可能创造出一种具有所需更高耐受性的新型酿酒酵母基因工程菌株，从而提高降解效率并降低成本。
