Synthetic biology focuses on the engineering and modular reengineering of various life systems, and has achieved some remarkable results in the fields of energy and medicine. Filamentous microorganisms (fungi, actinomycete, etc.) are the main source of secondary metabolites, and about 90% of known antibiotics are produced by such microorganisms, of which more than two-thirds are actinomyces represented by Streptomyces. Produced by bacteria. The prokaryotic streptococci genome is even larger than the eukaryotic yeast, and there are more genes, of which two-thirds of genes are involved in the regulation process. However, due to the mycelial growth characteristics of Streptomyces, it is difficult to precisely design, quantify and predict gene expression and become a huge technical bottleneck for the development of synthetic biology in this field. The "973" project "Study on Adaptation of Synthetic Microorganism Systems" by the researcher Zhang Lixin of the Institute of Microbiology, Chinese Academy of Sciences, is trying to solve this scientific problem, raise the level of understanding of the life cycle of Streptomyces, and realize the life of Streptomyces. Quantitative prediction, precision design, standard synthesis and precise control of the process.
Recently, the Institute of Microbiology Qi Chunbo and Zhang Lixin's research group have for the first time established a single cell precision quantification method based on flow cytometry and reporter gene (sfGFP) for Streptomyces biocomponents. The method optimizes the protoplast preparation scheme, replaces the sucrose buffer system that damages the flow cytometer, and simultaneously binds PI staining to separate dead cell individuals, thereby solving the defect of filamentous microbe growth morphological differentiation and programmed death that is not conducive to quantification. , which improves the measurement accuracy. After high-throughput portrayal of promoters and RBS and other regulatory elements with sufficiently large coverage, RiboJ insulators were successfully used to eliminate the interference effect of promoter and RBS in Streptomyces and to improve the modularity of these components. These regulatory element modules have also been successfully applied to activate the expression of the silent gene cluster lycopene in Streptomyces avermitilis, and through the predictable replacement of regulatory elements, the yield and efficiency of lycopene have been greatly improved, and the requirements for industrial production have been met. This work lays an important foundation for the application of synthetic biology to transform secondary metabolites of filamentous microorganisms and activate silent gene clusters.
The above findings were published on September 14th at Proceedings of the National Academy of Sciences USA. Zhang Lixin's research group Bai Chaoxian and Zhang Chunbo’s doctoral student Zhang Yang and assistant researcher Zhao Xuejin are the co-first authors of the paper. Researchers Zhang Lixin and Zhai Chunbo are the authors of the correspondence. Other authors include Hu Yiling, Xiang Sihai, and Miao Wei. The study was funded by the "973" project and the National Natural Science Foundation project.
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