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Abstract:
BackgroundThioesters of coenzyme A participate in 5% of all enzymatic
reactions. 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 of these metabolites explains the high
interest to trace and quantify them in microbial cells.ResultsHere, 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 assessed intracellular CoA thioesters down to the picomolar
level and exhibited high precision and reproducibility for all microbes,
as shown by principal component analysis. Furthermore, it provided
interesting insights into microbial CoA metabolism. A succinyl-CoA
synthase defective mutant of C. glutamicum exhibited an unaffected level
of succinyl-CoA that indicated a complete compensation by 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 actinomycete S. albus. The microbe
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.ConclusionsThe 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 one single protocol
promises to facilitate automatized efforts, which rely on standardized
workflows.
