A common approach for absolute quantification of short chain CoA thioesters in industrially relevant gram-positive and gram-negative prokaryotic and eukaryotic microbes

Abstract

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.

Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development

“…We hypothesized that the increased bottromycin titers could result from a higher precursor availability, as recently shown for other natural products (Gläser et al, 2020;Kuhl et al, 2020;Ser et al, 2016;Tang et al, 1994;Thykaer et al, 2010). Therefore, we analyzed the level of intracellular amino acids during growth (12 h, Figure 4b) and production (48 h, Figure 4c), including the building blocks of mature bottromycin A2: glycine, L-proline, L-valine, L-phenylalanine, L-aspartate, and L-cysteine (highlighted in blue in Figure 4b,c).…”

“…Cells were collected (10,000× g , 4°C, 10 min), washed twice with 15 ml deionized water, and freeze‐dried (Kuhl et al, 2020). Subsequently, the dry biomass was gravimetrically determined (Gläser et al, 2020). In microparticle cultivations of S. lividans , biomass data were corrected for the amount of talc added (Kuhl et al, 2020)…”

Microparticles globally reprogram Streptomyces albus toward accelerated morphogenesis, streamlined carbon core metabolism, and enhanced production of the antituberculosis polyketide pamamycin

“…Cells were collected (10,000 g , 4°C, 10 min), washed twice with 15 ml deionized water and freeze‐dried. Subsequently, the CDW was gravimetrically determined (Gläser et al, 2020). In microparticle cultivations, the measurements were corrected for the added talc (Driouch, Sommer, et al, 2010).…”

“…The analysis of CoA‐esters was conducted as recently described (Gläser et al, 2020). In short, a broth sample (8 mg CDW) was transferred into a precooled tube, which contained quenching and extraction solution (95% acetonitrile, 25 mM formic acid, −20°C) at a volume ratio of 1:4 followed by repetitive mixing and cooling on ice for 10 min, clarification from cell debris (15,000 g , 4°C, 10 min) and the addition of 10 ml supercooled deionized water.…”