文献类型：期刊文献

英文题名：Engineering of succinyl-CoA metabolism in view of succinylation regulation to improve the erythromycin production

作者：Ke, Xiang[1];Jiang, Xing[1];Huang, Mingzhi[1];Tian, Xiwei[1];Chu, Ju[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2022

卷号：106

期号：13-16

起止页码：5153

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20222912377029);WOS:【SCI-EXPANDED(收录号:WOS:000823351000003)】；

基金：This work was financially supported by the National Key Research Development Program of China (No. 2019YFA0904800), the National Natural Science Foundation of China (No. 21276081), and the National Key Research and Development Program of China (No.2019YFA0904300).

语种：英文

外文关键词：Saccharopolyspora erythraea; Succinylation; Erythromycin; Aconitase; CRISPRi

摘要：As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3 Delta sucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3 Delta sucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sgl was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea.