Huang Ming-Yue, Wang Wei-Yang, Liang Zhen-Zhen, Huang Yu-Chen, Yi Yi, Niu Fu-Xing
Guangxi Key Laboratory of Green Processing of Sugar Resources, Guangxi University of Science and Technology, Liuzhou 545006, China.
Department of Basic Medicine, Guangxi University of Science and Technology, Liuzhou 545006, China.
Biology (Basel). 2022 Oct 10;11(10):1484. doi: 10.3390/biology11101484.
Tolerance breeding through genetic engineering, sequence and omics analyses, and gene identification processes are widely used to synthesize biofuels. The majority of related mechanisms have been shown to yield endogenous genes with high expression. However, the process was time-consuming and labor-intensive, meaning there is a need to address the problems associated with the low-throughput screening method and significant time and money consumption. In this study, a combination of the limit screening method (LMS method) and product-tolerance engineering was proposed and applied. The MG1655 genomic DNA library was constructed using the shotgun method. Then, the cultures were incubated at concentrations of 0.25%, 0.5%, 0.75% and 1.0% of pinene with different inhibitory effects. Finally, the genes , , and were found to be associated with the enhanced tolerance of to pinene. Using the I-SceI cleavage system, the promoters of , and genes were replaced with P37. The final strain increased the production of pinene from glucose by 2.1 times.
通过基因工程、序列和组学分析以及基因鉴定过程进行耐受性育种被广泛用于合成生物燃料。大多数相关机制已被证明能产生高表达的内源基因。然而，该过程耗时且费力，这意味着有必要解决与低通量筛选方法以及大量时间和金钱消耗相关的问题。在本研究中，提出并应用了极限筛选法（LMS法）和产物耐受性工程的组合。使用鸟枪法构建了MG1655基因组DNA文库。然后，将培养物在具有不同抑制作用的0.25%、0.5%、0.75%和1.0%的蒎烯浓度下孵育。最后，发现基因、、和与增强对蒎烯的耐受性有关。使用I-SceI切割系统，将基因、和的启动子替换为P37。最终菌株使葡萄糖产生蒎烯的产量提高了2.1倍。
