Ren Junle, Zhang Miaomiao, Guo Xiaopeng, Zhou Xiang, Ding Nan, Lei Cairong, Jia Chenglin, Wang Yajuan, Zhao Jingru, Dong Ziyi, Lu Dong
Institute of Modern Physics, Chinese Academy of Sciences, No. 509 Nanchang Road, Lanzhou, 730000, Gansu, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Biotechnol Biofuels Bioprod. 2024 Aug 22;17(1):117. doi: 10.1186/s13068-024-02562-w.
Lignocellulose is a renewable and sustainable resource used to produce second-generation biofuel ethanol to cope with the resource and energy crisis. Furfural is the most toxic inhibitor of Saccharomyces cerevisiae cells produced during lignocellulose treatment, and can reduce the ability of S. cerevisiae to utilize lignocellulose, resulting in low bioethanol yield. In this study, multiple rounds of progressive ionizing radiation was combined with adaptive laboratory evolution to improve the furfural tolerance of S. cerevisiae and increase the yield of ethanol.
In this study, the strategy of multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution significantly improved the furfural tolerance of brewing yeast. After four rounds of experiments, four mutant strains resistant to high concentrations of furfural were obtained (SCF-R1, SCF-R2, SCF-R3, and SCF-R4), with furfural tolerance concentrations of 4.0, 4.2, 4.4, and 4.5 g/L, respectively. Among them, the mutant strain SCF-R4 obtained in the fourth round of radiation had a cellular malondialdehyde content of 49.11 nmol/mg after 3 h of furfural stress, a weakening trend in mitochondrial membrane potential collapse, a decrease in accumulated reactive oxygen species, and a cell death rate of 12.60%, showing better cell membrane integrity, stable mitochondrial function, and an improved ability to limit reactive oxygen species production compared to the other mutant strains and the wild-type strain. In a fermentation medium containing 3.5 g/L furfural, the growth lag phase of the SCF-R4 mutant strain was shortened, and its growth ability significantly improved. After 96 h of fermentation, the ethanol production of the mutant strain SCF-R4 was 1.86 times that of the wild-type, indicating that with an increase in the number of irradiation rounds, the furfural tolerance of the mutant strain SCF-R4 was effectively enhanced. In addition, through genome-transcriptome analysis, potential sites related to furfural detoxification were identified, including GAL7, MAE1, PDC6, HXT1, AUS1, and TPK3.
These results indicate that multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution is an effective mutagenic strategy for obtaining furfural-tolerant mutants and that it has the potential to tap genes related to the furfural detoxification mechanism.
木质纤维素是一种可再生且可持续的资源，用于生产第二代生物燃料乙醇以应对资源和能源危机。糠醛是木质纤维素处理过程中产生的对酿酒酵母细胞毒性最大的抑制剂，会降低酿酒酵母利用木质纤维素的能力，导致生物乙醇产量较低。在本研究中，将多轮渐进式电离辐射与适应性实验室进化相结合，以提高酿酒酵母对糠醛的耐受性并提高乙醇产量。
在本研究中，多轮渐进式X射线辐射与适应性实验室进化相结合的策略显著提高了酿酒酵母对糠醛的耐受性。经过四轮实验，获得了四株对高浓度糠醛具有抗性的突变菌株（SCF-R1、SCF-R2、SCF-R3和SCF-R4），其糠醛耐受浓度分别为4.0、4.2、4.4和4.5 g/L。其中，在第四轮辐射中获得的突变菌株SCF-R4在糠醛胁迫3小时后细胞丙二醛含量为49.11 nmol/mg，线粒体膜电位崩溃的减弱趋势明显，活性氧积累减少，细胞死亡率为12.60%，与其他突变菌株和野生型菌株相比，表现出更好的细胞膜完整性、稳定的线粒体功能以及限制活性氧产生的能力得到改善。在含有3.5 g/L糠醛的发酵培养基中，SCF-R4突变菌株的生长延迟期缩短，其生长能力显著提高。发酵96小时后，突变菌株SCF-R4的乙醇产量是野生型的1.86倍，表明随着辐照轮数的增加，突变菌株SCF-R4对糠醛的耐受性得到有效增强。此外，通过基因组-转录组分析，鉴定出了与糠醛解毒相关的潜在位点，包括GAL7、MAE1、PDC6、HXT1、AUS1和TPK3。
这些结果表明，多轮渐进式X射线辐射与适应性实验室进化相结合是获得糠醛耐受突变体的有效诱变策略，并且有挖掘与糠醛解毒机制相关基因的潜力。
