Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens
Rewiring the native methanol assimilation metabolism by incorporating the heterologous ribulose monophosphate cycle into Methylorubrum extorquens
复制标题
通过将异源核酮糖单磷酸循环纳入甲基红红菌中，重新连接天然甲醇同化代谢
DOI：
10.1016/j.ymben.2021.01.009
复制
发表时间：
2021-02-05
影响因子：
8.4
通讯作者：
Yang, Song
中科院分区：
文献类型：
--
作者：
Yuan, Xiao-Jie;Chen, Wen-Jing;Yang, Song
甲醇通过丝氨酸循环被同化以产生乙酰辅酶A而没有碳损失。然而，高活性丝氨酸循环需要高消耗的还原当量和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，基因组规模的通量平衡分析（FBA）预测，异源核酮糖单磷酸（RuMP）循环，一个更节能的甲醇同化途径的引入，理论上可以增加31.3%的模型alphaproteobacteria甲基营养型扭脱甲基红菌AM 1的生长速率。在此基础上，我们构建了一个新的协同同化途径，在体内纳入RuMP循环到M。内源性丝氨酸循环的外源性代谢。我们证明，协同途径的操作可以提高细胞生长速率16.5%和甲醇消耗速率13.1%。这种策略通过调节核心基因转录来重新连接中央甲基营养代谢，导致C2至C5中央中间体的池大小增加1.2至3.6倍，NADPH辅因子改善1.3倍。3-羟基丙酸（3-HP）是新设计的M.在摇瓶培养中，扭脱体AM 1的产酶量增加到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。3-HP的最终滴度在流加式生物反应器中显著提高至0.857 g/L，这与以甲烷和CO2为C1源的其他3-HP生产者相比更具竞争力。总的来说，我们目前的研究表明，工程协同甲醇同化途径是一个有前途的战略，以增加碳同化和减少化学品的产量在不同的宿主菌株C1微生物细胞工厂。
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.