Liuzzi Anna D, Tompkins Hannah L, Pallett Sarah K, Webster Lee, Mukamolova Galina V, Gregory Matthew A, Sim Martin, O'Hare Helen M
Department of Respiratory Sciences, University of Leicester, University Road, Leicester LE1 7RH, UK.
Isomerase Therapeutics, Newnham Building, Chesterford Research Park, Little Chesterford, Saffron Walden, Cambridge, Cambridgeshire, CB10 1XL, UK.
Microbiology (Reading). 2025 Aug;171(8). doi: 10.1099/mic.0.001583.
Actinobacteria are important for industrial production of antibiotics, fine chemicals and food and a source of new compounds for drug discovery. Their central metabolism is regulated by a conserved protein GarA that is unique to the Actinobacteria and has been studied in and . GarA regulates the TCA cycle and glutamate metabolism by direct binding to enzymes to modulate their activity on glutamate and alpha-ketoglutarate. Given the importance of the TCA cycle in the synthesis of acyl-CoA precursors for antibiotic biosynthesis, and increasing evidence for the role of nitrogen regulators in control of secondary metabolism, we hypothesized that engineering GarA could be used to enhance production of valuable metabolites. His-tagged GarA was introduced into , an overproducer of the polyketide antibiotic erythromycin. Phosphorylation of GarA was detected at the N-terminal ETTS motif, suggesting that it is regulated by protein kinases like in . GarA expression was observed at all growth stages, and a truncated form lacking the phosphorylation site accumulated during late fermentation. Engineered expressing phosphoablative GarA produced twofold more erythromycin, both in standard fermentation broth and in minimal medium. To investigate the mechanism for the increased titre, the engineered strain was characterized for transcription of erythromycin biosynthetic genes, as well as its ability to metabolize glutamate and its intracellular and extracellular aa content. The observed alterations in aa metabolism are consistent with the role of GarA as a TCA cycle regulator that may influence precursor supply for polyketide biosynthesis.
放线菌对于抗生素、精细化学品和食品的工业生产非常重要，也是药物发现中新化合物的来源。它们的中心代谢由一种保守蛋白GarA调节，GarA是放线菌特有的，并且已经在[具体研究对象1]和[具体研究对象2]中进行了研究。GarA通过直接结合酶来调节其对谷氨酸和α-酮戊二酸的活性，从而调控三羧酸循环（TCA循环）和谷氨酸代谢。鉴于TCA循环在抗生素生物合成的酰基辅酶A前体合成中的重要性，以及越来越多的证据表明氮调节因子在次级代谢控制中的作用，我们推测改造GarA可用于提高有价值代谢物的产量。将带有His标签的GarA导入聚酮类抗生素红霉素的高产菌株[具体菌株名称]中。在N端ETTS基序处检测到GarA的磷酸化，这表明它像在[具体参照对象]中一样受蛋白激酶调节。在所有生长阶段均观察到GarA的表达，并且在发酵后期积累了一种缺少磷酸化位点的截短形式。在标准发酵 broth和基本培养基中，表达磷酸化缺失型GarA的工程菌株产生的红霉素都增加了两倍。为了研究产量增加的机制，对工程菌株进行了红霉素生物合成基因转录的表征，以及其代谢谷氨酸的能力及其细胞内和细胞外氨基酸含量的测定。观察到的氨基酸代谢变化与GarA作为TCA循环调节因子的作用一致，该调节因子可能影响聚酮生物合成的前体供应。
