A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2022-12-01 DOI:10.1016/j.mec.2022.e00205
Andrea Steinmann , Katrin Schullehner , Anna Kohl , Christina Dickmeis , Maurice Finger , Georg Hubmann , Guido Jach , Ulrich Commandeur , Marco Girhard , Vlada B. Urlacher , Stephan Lütz
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Abstract

Microbial synthesis of monolignols and lignans from simple substrates is a promising alternative to plant extraction. Bottlenecks and byproduct formation during heterologous production require targeted metabolomics tools for pathway optimization.

In contrast to available fractional methods, we established a comprehensive targeted metabolomics method. It enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. Several cell disruption methods were compared. Hot water extraction was best suited regarding monolignol and lignan stability as well as extraction efficacy. The method was applied to compare enzymes for alleviating bottlenecks during heterologous monolignol and lignan production in E. coli. Variants of tyrosine ammonia-lyase had a considerable influence on titers of subsequent metabolites. The choice of multicopper oxidase greatly affected the accumulation of lignans. Metabolite titers were monitored during batch fermentation of either monolignol or lignan-producing recombinant E. coli strains, demonstrating the dynamic accumulation of metabolites.

The new method enables efficient time-resolved targeted metabolomics of monolignol- and lignan-producing E. coli. It facilitates bottleneck identification and byproduct quantification, making it a valuable tool for further pathway engineering studies. This method will benefit the bioprocess development of biotransformation or fermentation approaches for microbial lignan production.

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一种用于细胞外和细胞内代谢物定量的靶向代谢组学方法,涵盖了完整的单脂素和木脂素合成途径
微生物从简单底物中合成单脂醇和木脂素是一种很有前途的植物提取替代方法。异种生产过程中的瓶颈和副产物形成需要靶向代谢组学工具进行途径优化。与现有的分数方法相比,我们建立了一种全面的靶向代谢组学方法。它能够定量17个单脂素和木脂素途径的细胞外和细胞内代谢物,范围从氨基酸到pluviatolide。比较了几种细胞破坏方法。单脂素和木脂素的稳定性和提取效果最好的是热水提取。该方法用于比较缓解大肠杆菌中异源单脂素和木脂素生产瓶颈的酶。酪氨酸解氨酶的变异对随后代谢产物的滴度有相当大的影响。多铜氧化酶的选择对木脂素的积累有很大影响。在分批发酵过程中,对产生单脂素或木脂素的重组大肠杆菌菌株的代谢物滴度进行监测,显示代谢物的动态积累。新方法能够有效的时间分辨靶向代谢组学的单脂素和木脂素生产大肠杆菌。它有助于瓶颈识别和副产物量化,使其成为进一步途径工程研究的有价值的工具。该方法将有利于微生物木脂素生产的生物转化或发酵方法的生物工艺发展。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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