Journal
SYNTHETIC AND SYSTEMS BIOTECHNOLOGY
Volume 7, Issue 2, Pages 756-764Publisher
KEAI PUBLISHING LTD
DOI: 10.1016/j.synbio.2022.03.002
Keywords
TDP-L-mycarose; Glycosylated products; 3-O-alpha-mycarosylerythronolide B; Metabolic engineering; CRISPRi
Categories
Funding
- National Key R&D Program of China [2018YFA0900600]
- Program of Shanghai Academic Research Leader [20XD1404400]
- Strategic Priority Research Program Molecular mechanism of Plant Growth and Development of CAS [XDB27020202]
- National Natural Science Foundation of China [31670099]
- Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Science [ZSYS-016]
- International Partnership Program of Chinese Academy of Science [153D31KYSB20170121]
- National Key Laboratory of Plant Molecular Genetics, SIPPE, CAS
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In this study, we successfully biosynthesized 3-O-alpha-mycarosylerythronolide B (MEB) in Escherichia coli and improved its production using various metabolic engineering strategies. By blocking precursor glucose-1-phosphate competing pathways and overexpressing rfbA and rfbB, the MEB production was significantly increased. Furthermore, the implementation of CRISPRi to repress glycolysis and TDP-L-rhamnose pathway promoted TDP-L-mycarose biosynthesis. This study provides a promising platform for efficient production of erythromycins and other glycosylated products with unusual sugars.
The insufficient supply of sugar units is the key limitation for the biosynthesis of glycosylated products. The unusual sugar TDP-L-mycarose is initially attached to the C3 of the polyketide erythronolide B, resulting in 3-O- alpha-mycarosylerythronolide B (MEB). Here, we present the de novo biosynthesis of MEB in Escherichia coli and improve its production using multi-strategy metabolic engineering. Firstly, by blocking precursor glucose-1-phosphate competing pathways, the MEB titer of triple knockout strain QC13 was significantly enhanced to 41.2 mg/L, 9.8-fold to that produced by parental strain BAP230. Subsequently, the MEB production was further increased to 48.3 mg/L through overexpression of rfbA and rfbB. Moreover, the CRISPRi was implemented to promote the TDP-L-mycarose biosynthesis via repressing the glycolysis and TDP-L-rhamnose pathway. Our study paves the way for efficient production of erythromycins in E. coli and provides a promising platform that can be applied for biosynthesis of other glycosylated products with unusual sugars.
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