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. 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. 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.

中文翻译：

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原核和真核微生物中短链 CoA 硫酯绝对定量的常用方法。


辅酶 A 的硫酯参与所有酶促反应的 5%。在微生物细胞工厂中，它们充当具有公认商业价值的产品的构建模块，包括聚酮化合物、多不饱和脂肪酸、生物燃料和生物聚合物等天然产物。根据微生物的基因组库推断，许多微生物中存在大约 5-10 个短链硫酯的核心谱。这些代谢物的相关性解释了人们对在微生物细胞中追踪和量化它们的高度兴趣。在这里，我们描述了不同革兰氏阳性和革兰氏阴性细菌和真核酵母（即谷氨酸棒杆菌、白色链霉菌、恶臭假单胞菌和解脂耶氏酵母）中短链 CoA 硫酯的提取和绝对定量的常见工作流程。该方法对细胞内 CoA 硫酯的评估低至皮摩尔水平，并且如主成分分析所示，对所有微生物都表现出高精度和重现性。此外，它还为微生物 CoA 代谢提供了有趣的见解。谷氨酸棒杆菌的琥珀酰辅酶A合酶缺陷突变体表现出不受影响的琥珀酰辅酶A水平，这表明L-赖氨酸途径完全补偿以绕过中断的TCA循环。甲基丙二酸单酰辅酶 A 是高价值聚酮化合物的重要组成部分，被鉴定为放线菌 S. albus 中的主要辅酶 A 硫酯。该微生物的细胞内 CoA 硫酯丰度存在 10,000 倍以上的差异。白色沙门氏菌的重组菌株产生不同的抗结核聚酮帕马霉素衍生物，显示乙基丙二酰辅酶 A 途径的辅酶 A 硫酯显着减少，从而影响产物水平和谱。 短链 CoA 硫酯与合成工业产品的高度相关性以及从本工作中展示的示例中获得的有趣见解，表明比迄今为止更常规地分析微生物细胞工厂中的这些代谢物。由于其广泛的应用范围，所开发的方法似乎可用于此目的。因此，使用单一协议的可能性有望促进依赖标准化工作流程的自动化工作。