The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, Jülich, Germany.
Microb Biotechnol. 2024 Nov;17(11):e70044. doi: 10.1111/1751-7915.70044.
Malonyl-coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value-added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl-CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome-reduced Pseudomonas putida strain to increase acetyl-CoA carboxylation while limiting malonyl-CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked-out in specific combinations towards increasing the malonyl-CoA pool. An enzyme-coupled biosensor, based on the rppA gene, was employed to monitor malonyl-CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl-CoA into flaviolin, a red-colored polyketide. We isolated strains displaying enhanced malonyl-CoA availability via a colorimetric screening method based on the RppA-dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl-CoA levels. We further modified these strains by adding a non-canonical pathway that uses malonyl-CoA as precursor for poly(3-hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl-CoA-dependent pathways in P. putida.
丙二酰辅酶 A（CoA）是微生物细胞工厂合成多种增值化合物的关键前体，包括聚酮化合物、羧酸、生物燃料和聚羟基烷酸酯。由于其作为代谢枢纽的作用，丙二酰辅酶 A 的可用性受到几种必需代谢途径竞争的限制。为了解决这个限制，我们对一种基因组简化的假单胞菌进行了修饰，以增加乙酰辅酶 A 羧化作用，同时限制丙二酰辅酶 A 的利用。通过特定的组合敲除参与糖分解代谢及其调节、三羧酸（TCA）循环和脂肪酸生物合成的基因，以增加丙二酰辅酶 A 池。我们采用基于 rppA 基因的酶偶联生物传感器在体内监测丙二酰辅酶 A 水平。RppA 是一种 III 型聚酮合酶，将丙二酰辅酶 A 转化为 flaviolin，一种红色的聚酮化合物。我们通过基于 RppA 依赖性红色色素沉着的比色筛选方法分离出显示增强丙二酰辅酶 A 可用性的菌株；直接 flaviolin 定量确定了四个工程菌株的丙二酰辅酶 A 水平有显著增加。我们进一步通过添加非经典途径修饰这些菌株，该途径将丙二酰辅酶 A 用作聚（3-羟基丁酸酯）生物合成的前体。这些操作导致完全工程化菌株中聚合物积累增加，验证了我们在假单胞菌中增强丙二酰辅酶 A 依赖性途径产量的一般策略。
