Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply
Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply
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DOI：
10.1016/j.ymben.2016.11.010
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发表时间：
2017-01-01
影响因子：
8.4
通讯作者：
Xing, Xin-Hui
中科院分区：
文献类型：
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作者：
Liang, Wei-Fan;Cui, Lan-Yu;Xing, Xin-Hui
乙酰辅酶A不仅是细胞重要的中间代谢产物，而且是生产工业上感兴趣的代谢产物的重要前体。扭脱甲基杆菌AM 1是利用甲醇作为碳源的甲基营养型细胞工厂的模式菌株，由于其产生丰富的辅酶A化合物，能够指导从甲醇合成不同的有用化合物，因此受到关注。然而，乙酰辅酶A并不总是有效地积累在M。extorquens AM 1，因为它位于三个循环中心代谢途径的中心。在这里，我们成功地证明了传感器辅助转录调节因子工程（SATRE）的策略，以控制代谢通量的重新分布，以增加来自甲醇的乙酰辅酶A通量，用于M.通过引入甲羟戊酸合成途径对扭脱醌AM 1进行改造。甲羟戊酸生物传感器的构建，我们成功地分离出突变株（Q49）的甲羟戊酸浓度增加60%（乙酰辅酶A衍生产品）后，基于传感器的高通量筛选的QscR转录调控文库。突变的QscR-49调节子（Q8*、T61 S、N72 Y、E160 V）失去了N-末端α-螺旋，并经历了C末端RD-I结构域二级结构的变化，这两个区域与其与DNA的相互作用有关。C-13标记分析表明，乙酰辅酶A通量提高了7%，转录分析表明，QscR具有全局效应，两个关键点，NADPH的产生和fumC的过表达，可能有助于碳通量的重新分配。QscR-49突变株在5L生物反应器中的补料分批发酵产生了2.67g/L的甲羟戊酸浓度，这相当于0.055mol乙酰辅酶A/mol甲醇的总产量，这是M. extorquens AM1.这项工作是第一次尝试调节M。在转录水平上对extroquens AM 1的功能进行了初步研究，为阐明碳通量调控的分子机制提供了理论依据。
Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal a-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. C-13 labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67 g/L, which was equivalent to an overall yield of 0.055 mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.