A common approach for absolute quantification of short chain CoA thioesters in prokaryotic and eukaryotic microbes

Abstract

Background: Thioesters 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. 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 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. 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 one single protocol promises to facilitate automatized efforts, which rely on standardized workflows.

GC/MS-based 13C metabolic flux analysis resolves the parallel and cyclic photomixotrophic metabolism of Synechocystis sp. PCC 6803 and selected deletion mutants including the Entner-Doudoroff and phosphoketolase pathways

“…Metabolite extraction and sampling was performed as described previously [ 89 ]. Prior to extraction, cells were quenched in − 20 °C buffer [25 mM formic acid, 95% (v/v) acetonitrile in water] at a volume ratio of 1–3.…”

Structure, Biosynthesis, and Biological Activity of Succinylated Forms of Bacteriocin BacSp222

“…Effective succinylation of proteins by suc-CoA is a further consequence of the high level of this metabolite in cells (100-600 nM in eukaryotic mitochondria and ca. 100 nmol g −1 in bacterial cells) [7,9]. The level of succinylated proteins in certain cells results from the equilibrium between the activity of enzymes catalysing suc-CoA formation and enzymes that desuccinylate proteins.…”

Channelling carbon flux through the meta‐cleavage route for improved poly(3‐hydroxyalkanoate) production from benzoate and lignin‐based aromatics in Pseudomonas putida H

“…Sampling, sample processing and analysis of intracellular CoA thioesters were conducted as described recently for the related strain P. putida KT2440, involving simultaneous quenching and extraction (95% acetonitrile, 25 mM formic acid, −20 °C), sample clean up, CoA ester analysis by a triple quadrupole MS (QTRAP 6500+; AB Sciex, Darmstadt, Germany) coupled to an HPLC system (Agilent Infinity 1290 System; Agilent Waldbronn, Waldbronn, Germany) and absolute quantification against an internal 13 C enriched standard (Gläser et al ., 2020 ).…”