Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides
| Autor: | Lu Zhang, Gao-Yi Tan, Dongyuan Lv, Haihong Chen, Lixin Zhang, Yue Jiang, Ke-Feng Wang, Chuan Li, Liming Zhou, Dan Li, Xinwei He, Mindong Liang, Tong Shi, Biqin Chen, Zhiheng Yang, Jing Liu, Weishan Wang |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Reporter gene
biology Strain (chemistry) QH301-705.5 Chemistry Biomedical Engineering Promoter Rhodobacter sphaeroides Promoter library biology.organism_classification Co-enzyme Q10 Applied Microbiology and Biotechnology Green fluorescent protein Transcriptome Synthetic biology Red fluorescent protein Biochemistry Structural Biology Genetics Biology (General) Transcriptomics Gene TP248.13-248.65 Biotechnology |
| Zdroj: | Synthetic and Systems Biotechnology, Vol 6, Iss 4, Pp 335-342 (2021) |
| ISSN: | 2405-805X |
| DOI: | 10.1016/j.synbio.2021.09.011 |
| Popis: | The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, Prsp_7571 and Prsp_6124, showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a Prsp_7571-derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs. |
| Databáze: | OpenAIRE |
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