Qingdao Energy Institute successfully developed the cyanobacterial supermutation system

Recently, the scientific research team of Lv Xuefeng of the Microbial Manufacturing Engineering Center of Qingdao Institute of Bioenergy and Process Research, Chinese Academy of Sciences developed a new cyanobacterial supermutation system, which breaks through the limitation of high fidelity of cell genome replication on its evolutionary rate, greatly improves the replication mutation rate and adaptive evolution rate of Synococcus cells through genetic and environmental synergistic perturbations, successfully obtains evolutionary algal strains with significantly improved high temperature and high light tolerance, and reveals the key targets and functional mechanisms affecting the high temperature and high light tolerance of cyanobacteria. The results were published online March 4 in Nature Communications.

Synergistic perturbations of genetics and environment stimulate the supermutation state of Synococcus Photocourtesy of Qingdao Institute of Bioenergy and Processes

Photosynthesis is the most important biochemical process on Earth, and the photosynthetic carbon sequestration activities of plants and algae provide the most fundamental primary productivity for the maintenance and development of the biosphere. Improving the high temperature and high light tolerance of photosynthetic organisms is an important direction of photosynthesis research.

Cyanobacteria is a model system for studying photosynthesis, improving the high temperature and high phototolerance of cyanobacteria and analyzing its functional mechanism, which has guiding and demonstration value for the optimization of other photosynthetic biological systems. However, the damage mechanism of high temperature and high light stress on cyanobacteria has not been clearly analyzed, and rational metabolic engineering strategies are difficult to effectively improve the tolerance of cells at high temperature and high light, and evolutionary engineering is an effective means to modify such complex physiological phenotypes.

Using PCC 7942, the cyanobacterial model algae strain Synechococcus 7942, the research team systematically identified key genes affecting its genome replication fidelity, and increased the replication mutation rate of recombinant algae strains by two orders of magnitude through the integration strategy of fidelity element knockout-mutagenic element expression.

On this basis, the research team found that environmental stress can also affect the mutation rate of Synococcus, and the coupling of genetic fidelity mechanism defects with culture environmental stress can trigger the supermutation state, increasing the cell mutation rate by three orders of magnitude.

Based on the above results, the research team proposed a synergistic mechanism between environmental stress and replication fidelity mechanism defects, and used the system to optimize the high temperature and high light tolerance of Synococcus.

Using the above supermutation system, the research team successfully obtained the evolutionary algae strain of Synococcus with greatly improved high temperature and high light tolerance within two weeks, which showed great advantages in efficiency compared with laboratory adaptive evolution and conventional chemical mutagenesis. Under high temperature and light conditions when the starting algae cannot survive, the Synchronococcus evolutionary algae strain shows good adaptability and rapid growth ability.

The research team performed whole genome sequencing on 23 obtained high-temperature and high-light tolerant algae strains, and combined the application of forward genetics and reverse genetics strategies, and identified the main mutations that confer high light tolerance to Syncoccus as FoF1-ATP synthetase alpha subunit C252A mutation and the NC2 mutation in the promoter region of shikimate kinase. Among them, the mechanism by which NC2 mutations lead to high temperature and high phototolerance of Synechococcus by increasing the expression level of shikimate kinase was reported for the first time. The research team verified the mechanism, and the overexpression of shikimate kinase effectively improved the growth stability of recombinant algae strains under high temperature and high light stress, indicating that this strategy may have wide applicability to different cyanobacterial strains.

The research team further analyzed the mechanism of NC2 mutation to improve the high temperature and high phototolerability of Synococcus by upregulating the expression of shikimonic acid kinase, and combined with the analysis of transcriptome, proteome and photosynthetic physiological parameters, it was found that the mutation caused significant changes in the photosynthetic and carbon sequestration system of Synococcus, reduced the excessive absorption of light energy, enhanced the cyclic electron flow and oxidative phosphorylation activity of cells, and enhanced glycogen and protein synthesis, ultimately ensuring an efficient and stable photosynthetic carbon sequestration process.

In this study, a novel cyanobacterial hypermutation system was developed and its effectiveness was confirmed by the modification of high temperature and high phototolerance of Synococcus integras, which provided a reliable tool for the optimization of complex photosynthetic physiological phenotypes. The phenomenon that the increase in the expression of shikhitate kinase in the study leads to the optimization of high temperature and high light tolerance ability of cyanobacteria, which enriches the understanding of photosynthetic physiological metabolism and provides new enlightenment for the artificial design of high-light efficiency cyanobacterial chassis in the future.

The research has been supported by the National Key R&D Program of China, the National Natural Science Foundation of China, the Chinese Academy of Sciences Young Talents Innovation Promotion Association, the Joint Fund of the Clean Energy Innovation Research Institute, and the Shandong Provincial Talent Program. (Source: China Science News, Liao Yang, Kong Fengru)

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