Methanol is assimilated through the serine cycle to generate acetyl-CoA without carbon loss. However, a highly active serine cycle requires high consumption of reducing equivalents and ATP, thereby leading to the impaired efficiency of methanol conversion to reduced chemicals. In the present study, a genome-scale flux balance analysis (FBA) predicted that the introduction of the heterologous ribulose monophosphate (RuMP) cycle, a more energy-efficient pathway for methanol assimilation, could theoretically increase growth rate by 31.3% for the model alphaproteobacterial methylotroph Methylorubrum extorquens AM1. Based on this analysis, we constructed a novel synergistic assimilation pathway in vivo by incorporating the RuMP cycle into M. extroquens metabolism with the intrinsic serine cycle. We demonstrated that the operation of the synergistic pathway could increase cell growth rate by 16.5% and methanol consumption rate by 13.1%. This strategy rewired the central methylotrophic metabolism through adjusting core gene transcription, leading to a pool size increase of C2 to C5 central intermediates by 1.2- to 3.6-fold and an NADPH cofactor improvement by 1.3-fold. The titer of 3-hydroxypropionic acid (3-HP), a model product in the newly engineered chassis of M. extorquens AM1, was increased to 91.2 mg/L in shake-flask culture, representing a 3.1-fold increase compared with the control strain with only the serine cycle. The final titer of 3-HP was significantly improved to 0.857 g/L in the fed-batch bioreactor, which was more competitive compared with the other 3-HP producers using methane and CO₂ as C1 sources. Collectively, our current study demonstrated that engineering the synergistic methanol assimilation pathway was a promising strategy to increase the carbon assimilation and the yields of reduced chemicals in diverse host strains for C1 microbial cell factories.

甲醇通过丝氨酸循环被同化以生成乙酰辅酶 A，而没有碳损失。然而，高度活跃的丝氨酸循环需要大量消耗还原当量和 ATP，从而导致甲醇转化为还原化学品的效率受损。在本研究中，基因组规模通量平衡分析 (FBA) 预测，引入异源单磷酸核酮糖 (RuMP) 循环，这是一种更节能的甲醇同化途径，理论上可以将模型的增长率提高 31.3% αproteobacterial甲基营养菌 Methylorubrum extorquens AM1。基于此分析，我们通过将 RuMP 循环整合到M. extroquens 中构建了一种新的体内协同同化途径内源丝氨酸循环代谢。我们证明了协同途径的运行可以使细胞生长率提高 16.5%，甲醇消耗率提高 13.1%。该策略通过调整核心基因转录来重新连接中央甲基营养代谢，导致 C2 到 C5 中央中间体的池大小增加 1.2 到 3.6 倍，NADPH 辅因子改善 1.3 倍。3-羟基丙酸 (3-HP) 的滴度，M. extorquens新设计的底盘中的模型产品AM1 在摇瓶培养中增加到 91.2 mg/L，与仅具有丝氨酸循环的对照菌株相比增加了 3.1 倍。在补料分批生物反应器中，3-HP 的最终滴度显着提高至 0.857 g/L，与其他使用甲烷和 CO ₂作为 C1 源的3-HP 生产商相比，这更具竞争力。总的来说，我们目前的研究表明，设计协同甲醇同化途径是一种很有前景的策略，可以在 C1 微生物细胞工厂的不同宿主菌株中增加碳同化和减少化学品的产量。