文献类型：期刊文献

英文题名：A dynamic model-based preparation of uniformly-13C-labeled internal standards facilitates quantitative metabolomics analysis of Penicillium chrysogenum

作者：Wang, Guan[1];Chu, Ju[1];Zhuang, Yingping[1];van Gulik, Walter[2];Noorman, Henk[3,4]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]Delft Univ Technol, Dept Biotechnol, Cell Syst Engn, Delft, Netherlands;[3]DSM Biotechnol Ctr, Delft, Netherlands;[4]Delft Univ Technol, Dept Biotechnol, Delft, Netherlands

年份：2019

卷号：299

起止页码：21

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20191806863549);WOS:【SCI-EXPANDED(收录号:WOS:000468162100004)】；

基金：This work was financially subsidized by the NWO-MoST Joint Program, Grant number, 2013DFG32630, the 111 Project (B18022), the National key research and development program, 2017ZX7402003, and the Fundamental Research Funds for the Central Universities, 22221818014.

语种：英文

外文关键词：Dynamic; Fed-batch model; Isotope dilution mass spectrometry (IDMS); Perturbation; Metabolomics; Penicillium chrysogenum

摘要：Targeted, quantitative metabolomics can, in principle, provide precise information on intracellular metabolite levels, which can be applied to accurate modeling of intracellular processes required in systems biology and metabolic engineering. However, quantitative metabolite profiling is often hampered by biased mass spectrometry-based analyses caused by matrix effects, the degradation of metabolites and metabolite leakage during sample preparation, and unexpected variation in instrument responses. Isotope Dilution Mass Spectrometry (IDMS) has been proven as the most accurate method for high-throughput detection of intracellular metabolite concentrations, and the key has been the acquisition of the corresponding fully uniformly (U)-C-13-labeled metabolites to be measured. Here, we have prepared U-C-13-labeled cell extracts by cultivating P. chrysogenum in a fed-batch fermentation with fully U-C-13-labeled substrates. Towards this goal, a dynamic fed-batch model describing P. chrysogenum growth and penicillin production was used to simulate the fermentation process and design the fed-batch fermentation media. Further, a case study with extensive intracellular metabolomics data from glucose-limited cultivation of Penicillium chrysogenum under both single and repetitive glucose pulses was illustrated by using the IDMS methods with the prepared U-C-13-labeled cell extracts as internal standards. In conclusion, the IDMS method can be incorporated into well-established fast sampling and quenching protocols to obtain dynamic quantitative in vivo metabolome data at the timescales of (tens of) seconds and elucidate the underlying regulatory architecture. The case study revealed gross differences between single and repeated pulses, which suggests that single pulse studies have limited value for understanding of metabolic responses in large-scale bioreactors. Instead, intermittent feeding should be favored.