| Popis: |
Background: Thioesters of coenzyme A participate in 5% of all enzymatic reactions and at least one third of all cellular carbon is typically metabolized through a CoA thioester. In microbial cell factories, they function as building blocks for products of recognized commercial value, including natural products such as polyketides, polyunsaturated fatty acids, biofuels, and biopolymers. A core spectrum of approximately 5 – 10 short chain thioesters is present in many microbes, as inferred from their genomic repertoire. The relevance these metabolites explains the high interest to trace and quantify them in microbial cells.Results: Here, we describe a common workflow for extraction and absolute quantification of short chain CoA thioesters in different gram-positive and gram-negative bacteria and eukaryotic yeast, i.e. Corynebacterium glutamicum, Streptomyces albus, Pseudomonas putida, and Yarrowia lipolytica. The approach detected CoA thioesters down to the level of 40 attomole and exhibited high precision and reproducibility for all microbes as shown by principal component analysis. Furthermore, it provided interesting insights into microbial CoA-spectra. A succinyl-CoA synthase defective mutant of C. glutamicum, exhibited an unaffected level of succinyl-CoA, which indicated a complete compensation of the l-lysine pathway to bypass the disrupted TCA cycle. Methylmalonyl-CoA, an important building block of high-value polyketides, was identified as dominant CoA thioester in the microbe. S. albus revealed a more than 10,000-fold difference in the abundance of intracellular CoA thioesters. A recombinant strain of S. albus, which produced different derivatives of the antituberculosis polyketide pamamycin, revealed a significant depletion of CoA thioesters of the ethylmalonyl CoA pathway, influencing product level and spectrum. Conclusions: The high relevance of short chain CoA thioesters to synthetize industrial products and the interesting insights gained from the examples shown in this work, suggest analyzing these metabolites in microbial cell factories more routinely than done so far. Due to its broad application range, the developed approach appears useful to be applied this purpose. Hereby, the possibility to use on single protocol promises to facilitate automatized efforts, which rely on standardized workflows. |
