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

Synthesis pathway of monolignol and lignan metabolites from phenylalanine/tyrosine to pluviatolide. Pathway sections highlighted in color were studied in vivo. The yellow section is included in E. coli strains type I, which produce coniferyl alcohol from tyrosine. E. coli strains type II express the genes necessary for the pathway section highlighted in blue, resulting in the production of secoisolariciresinol from coniferyl alcohol. PAL: phenylalanine ammonia-lyase; TAL: tyrosine ammonia-lyase; C4H: cinnamate 4-hydroxylase; 4HPA3H: 4-hydroxyphenylacetate 3-monooxygenase; COMT: caffeic acid O-methyltransferase; CCoAOMT: caffeoyl-CoA O-methyltransferase; 4CL: 4-coumarate:CoA ligase; CCR: cinnamoyl-CoA reductase; CAD: cinnamyl-alcohol dehydrogenase; –[H]: one-electron oxidation, for instance, catalyzed by laccase from Corynebacterium glutamicum (CgL1) or copper efflux oxidase (CueO); PLR: pinoresinol-lariciresinol reductase; SDH: secoisolariciresinol dehydrogenase; CYP: P450 monooxygenase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In the framework of the collaborative BMBF-funded project "LignaSyn" a comprehensive targeted metabolomics method was established, which enables the quantification of 17 extra- and intracellular metabolites of the monolignol and lignan pathway, ranging from amino acids to pluviatolide. The publication recently appeared in Metabolic Engineering Communications. Below you can read the abstract. The link to the whole publication can be found in the reference at the bottom.

"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."

Steinmann A, Schullehner K, Kohl A, Dickmeis C, Finger M, Hubmann G, Jach G, Commandeur U, Girhard M, Urlacher VB, Lütz S, 2022,  A targeted metabolomics method for extra- and intracellular metabolite quantification covering the complete monolignol and lignan synthesis pathway, Metabolic Engineering Communications, in press, https://doi.org/10.1016/j.mec.2022.e00205

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Kategorie/n: BC Urlacher
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