Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii
Phage serine integrase-mediated genome engineering for efficient expression of chemical biosynthetic pathway in gas-fermenting Clostridium ljungdahlii
复制标题
噬菌体丝氨酸整合酶介导的基因组工程在气体发酵永大梭菌中高效表达化学生物合成途径
DOI：
10.1016/j.ymben.2019.01.005
复制
发表时间：
2019-03-01
影响因子：
8.4
通讯作者：
Gu, Yang
中科院分区：
文献类型：
--
作者：
Huang, He;Chai, Changsheng;Gu, Yang
能够利用CO和CO2的气体发酵梭菌的真实的价值在于它们被开发成细胞工厂以生产各种大宗化学品和燃料的潜力。该过程需要异源化学生物合成途径的快速染色体整合，但由于缺乏能够在这些厌氧菌中进行有效基因组工程的遗传工具，这一过程受到阻碍。在这里，我们开发了噬菌体丝氨酸整合酶介导的位点特异性基因组工程技术在杨氏梭菌，主要产乙酸气体发酵微生物之一。将两种异源噬菌体附着/整合（Att/Int）系统（来自艰难梭菌和链霉菌）引入C. ljungdahlii，并被证明是高度活性的，通过单交换重组实现了整个供体载体的有效染色体整合。在此基础上，我们在CRISPR-Cas9编辑系统的辅助下，通过“双整合酶盒交换”（DICE）策略，进一步实现了靶DNA片段的无标记染色体整合。作为概念证明，将来自丙酮丁醇梭菌的丁酸生产途径整合到C.在不引入额外标记的情况下，将该途径基因导入扬氏菌基因组，从而能够稳定表达该途径基因。得到的工程菌株通过发酵合成气（CO2/CO）在3天内产生1.01g/L的丁酸。更重要的是，工程菌表现出良好的遗传稳定性，连续传代后仍保持丁酸生产能力。本研究开发的系统克服了现有遗传工具在C. ljungdahlii，并且可以扩展到其他梭菌属物种。
The real value of gas-fermenting clostridia, capable of using CO and CO2, resides in their potential of being developed into cell factories to produce various bulk chemicals and fuels. This process requires rapid chromosomal integration of heterologous chemical biosynthetic pathways, which is impeded by the absence of genetic tools competent for efficient genome engineering in these anaerobes. Here, we developed a phage serine integrase-mediated site-specific genome engineering technique in Clostridium ljungdahlii, one of the major acetogenic gas-fermenting microbes. Two heterologous phage attachment/integration (Att/Int) systems (from Clostridium difficile and Streptomyces) were introduced into C. ljungdahlii and proven to be highly active, achieving efficient chromosomal integration of a whole donor vector via single-crossover recombination. Based on this, we further realized markerless chromosomal integration of target DNA fragments through a "dual integrase cassette exchange" (DICE) strategy with the assistance of the CRISPR-Cas9 editing system. As a proof of concept, a butyric acid production pathway from Clostridium acetobutylicum was integrated into the C. ljungdahlii genome without the introduction of extra markers, enabling stable expression of the pathway genes. The resulting engineered strain produced 1.01 g/L of butyric acid within 3 days by fermenting synthesis gas (CO2/CO). More importantly, the engineered strain showed good genetic stability and maintained butyric acid production ability after continuous subculturing. The system developed in this study overcomes the deficiencies of currently available genetic tools in the chromosomal integration of large DNA fragments (rapid, markerless and stable) in C. ljungdahlii, and may be extended to other Clostridium species.