한빛사 논문
Hyun Aaron Kim1,7†, Hyun Ju Kim2†, Jihoon Park3, Ah Reum Choi4, Kyoo Heo6, Haeyoung Jeong5, Kwang.Hwan Jung4, Yeong.Jae Seok6, Pil Kim3* and Sang Jun Lee2*
1 Hana Academy Seoul, Seoul, Republic of Korea. 2 Department of Systems Biotechnology, Chung-Ang University, Anseong, Gyeonggi, Republic of Korea. 3 Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi, Republic of Korea. 4 Department of Life Sciences, Sogang University, Seoul, Republic of Korea. 5 Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea. 6 Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea. 7 Present Address: Department of Biological Sciences, Seoul National University, Seoul, Republic of Korea.
†Hyun Aaron Kim and Hyun Ju Kim contributed equally to this work.
*Correspondence: Pil Kim, Sang Jun Lee
Abstract
Background
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.
Results
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.
Conclusions
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.
Keywords : Adaptive laboratory evolution, Strain optimization, Chemotroph, Phototroph, Rhodopsin, Proton pumping
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