Kim Hyun Aaron, Kim Hyun Ju, Park Jihoon, Choi Ah Reum, Heo Kyoo, Jeong Haeyoung, Jung Kwang-Hwan, Seok Yeong-Jae, Kim Pil, Lee Sang Jun
Hana Academy Seoul, Seoul, Republic of Korea.
Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea.
Microb Cell Fact. 2017 Jun 15;16(1):111. doi: 10.1186/s12934-017-0725-6.
The expression of the Gloeobacter rhodopsin (GR) in a chemotrophic Escherichia coli enables the light-driven phototrophic energy generation. Adaptive laboratory evolution has been used for acquiring desired phenotype of microbial cells and for the elucidation of basic mechanism of molecular evolution. To develop an optimized strain for the artificially acquired phototrophic metabolism, an ancestral E. coli expressing GR was adaptively evolved in a chemostat reactor with constant illumination and limited glucose conditions. This study was emphasized at an unexpected genomic mutation contributed to the improvement of microbial performance.
During the chemostat culture, increase of cell size was observed, which were distinguished from that of the typical rod-shaped ancestral cells. A descendant ET5 strain was randomly isolated from the chemostat culture at 88-days. The phototrophic growth and the light-induced proton pumping of the ET5 strain were twofold and eightfold greater, respectively, than those of the ancestral E. coli strain. Single point mutation of C1082A at dgcQ gene (encoding diguanylate cyclase, also known as the yedQ gene) in the chromosome of ET5 strain was identified from whole genome sequencing analysis. An ancestral E. coli complemented with the same dgcQ mutation from the ET5 was repeated the subsequently enhancements of light-driven phototrophic growth and proton pumping. Intracellular c-di-GMP, the product of the diguanylate cyclase (dgcQ), of the descendant ET5 strain was suddenly increased while that of the ancestral strain was negligible.
Newly acquired phototrophic metabolism of E. coli was further improved via adaptive laboratory evolution by the rise of a point mutation on a transmembrane cell signaling protein followed by increase of signal molecule that eventually led an increase proton pumping and phototrophic growth.
嗜盐菌视紫红质（GR）在化能营养型大肠杆菌中的表达能够实现光驱动的光合能量产生。适应性实验室进化已被用于获得微生物细胞所需的表型以及阐明分子进化的基本机制。为了开发一种用于人工获得光合代谢的优化菌株，将表达GR的祖先大肠杆菌在恒化器反应器中进行适应性进化，条件为持续光照和有限的葡萄糖。本研究重点关注一个意外的基因组突变对微生物性能改善的作用。
在恒化器培养过程中，观察到细胞大小增加，这与典型的杆状祖先细胞不同。在第88天从恒化器培养物中随机分离出一个后代ET5菌株。ET5菌株的光合生长和光诱导质子泵浦分别比祖先大肠杆菌菌株高两倍和八倍。通过全基因组测序分析确定，ET5菌株染色体上的dgcQ基因（编码二鸟苷酸环化酶，也称为yedQ基因）发生了C1082A单点突变。用来自ET5的相同dgcQ突变互补的祖先大肠杆菌重复了随后光驱动光合生长和质子泵浦的增强。后代ET5菌株中二鸟苷酸环化酶（dgcQ）的产物细胞内c - 二鸟苷酸突然增加，而祖先菌株的则可忽略不计。
大肠杆菌新获得的光合代谢通过适应性实验室进化进一步改善，这是由于跨膜细胞信号蛋白上的一个点突变增加，随后信号分子增加，最终导致质子泵浦和光合生长增加。
