Jing Zhang: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Zilong He: Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Interdisciplinary Innovation Institute of Medicine and Engineering, Beihang University, Beijing, 100191, China.
JinTian Xu: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Shuting Song: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Qianhui Zhu: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Guoguo Wu: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Ying Guan: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Xiaonong Wu: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Rong Yue: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Yue Wang: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Tao Yu: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Songnian Hu: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Fuping Lu: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China.
Huitu Zhang: Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, China. hzhang@tust.edu.cn.
The biosynthesis of the valuable antibiotic enduracidin by Streptomyces fungicidicus TXX3120 is a complex multistep process. To identify the rate-limiting step of the entire biosynthetic process, we carried out a deep RNA sequencing towards the mycelia of TXX3120 at different fermentation stages. Comparative RNA-seq analysis indicated that the expression level of the endC gene during the enduracidin production phase was evidently lower than that of the other relevant genes to enduracidin biosynthesis. This result was further confirmed by quantitative RT-PCR, and the giant non-ribosomal peptide synthase (NRPS) encoded by endC was predicated to be the rate-limiting enzyme in enduracidin biosynthesis. To increase the expression of endC during the enduracidin production phase, a reporter-based selection system was developed by genetically replacing the initial part of the endC gene with a thiostrepton resistance gene (tsr), which will then act as a selectable marker to report the expression level of the rate-limiting gene endC, thereby facilitating the selection of enduracidin-overproducing mutants following random mutagenesis. After one round of mutagenesis, thiostrepton resistance selection, and restoration of the endC gene, three mutant strains with improved endC expression levels were obtained. Their highest enduracidin titers reached 9780.54, 9272.46, and 8849.06 U/mL, respectively representing 2.31-, 2.19-, and 2.09-fold of the initial industrial strain TXX3120. Our research provides a useful strategy for the rational breeding of industrial strains that synthesize complex natural products.
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Grants
81373309/National Natural Science Foundation of China
