We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP⁺-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases.

我们设计II型甲烷营养，Methylosinus弯菌通过异源表达重构丙二酰-CoA途径OB3b，3-羟基丙酸（3HP）生产Chloroflexus子囊菌丙二酰-CoA还原酶（MCR），一个双功能酶。设计并实施了两种策略来增加丙二酰辅酶A库，从而提高3HP产量。首先，我们通过过表达内源性乙酰基CoA羧化酶（ACC）来设计丙二酰CoA前体的供应，从而显着提高3HP的产量。生物素蛋白连接酶（BPL）和苹果酸酶（NADP ⁺-ME）导致ACC过表达细胞的3HP滴度分别增加了约22.7％和约34.5％。此外，重建了乙酰辅酶A羧化旁路路线，以提高3HP生产率。弗氏丙酸杆菌的甲基丙二酰辅酶A羧基转移酶（MMC）的共表达提供MMC前体的磷酸烯醇丙酮酸羧化酶（PEPC）进一步提高了3HP效价。与仅MCR过表达的菌株相比，在过表达MCR，MMC和PEPC的OB3b-MCRMP菌株中最高的3HP产量为49 mg / L，其滴度提高了2.4倍。最后，通过生物反应器操作，使用OB3b-MCRMP菌株，在42小时内可以得到60.59 mg / L的3HP，与烧瓶培养相比，其容积生产率提高了6.36倍。这项工作演示了II型甲烷营养生物的代谢工程，为使用II型甲烷营养生物作为生化生产以及减少温室气体的细胞工厂打开了大门。