Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2.,ACS Synthetic Biology

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Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2019-03-20 , DOI: 10.1021/acssynbio.8b00481
Timothy B Roth _{1,

2,

3} , Benjamin M Woolston ₄ , Gregory Stephanopoulos ₄ , David R Liu _{1,

2,

3}

Affiliation

Synthetic methylotrophy, the modification of organisms such as E. coli to grow on methanol, is a longstanding goal of metabolic engineering and synthetic biology. The poor kinetic properties of NAD-dependent methanol dehydrogenase, the first enzyme in most methanol assimilation pathways, limit pathway flux and present a formidable challenge to synthetic methylotrophy. To address this bottleneck, we used a formaldehyde biosensor to develop a phage-assisted noncontinuous evolution (PANCE) selection for variants of Bacillus methanolicus methanol dehydrogenase 2 (Bm Mdh2). Using this selection, we evolved Mdh2 variants with up to 3.5-fold improved Vmax. The mutations responsible for enhanced activity map to the predicted active site region homologous to that of type III iron-dependent alcohol dehydrogenases, suggesting a new critical region for future methanol dehydrogenase engineering strategies. Evolved Mdh2 variants enable twice as much 13C-methanol assimilation into central metabolites than previously reported state-of-the-art methanol dehydrogenases. This work provides improved Mdh2 variants and establishes a laboratory evolution approach for metabolic pathways in bacterial cells.

中文翻译：

噬菌体辅助的甲醇芽孢杆菌甲醇脱氢酶2。

合成甲基营养素是对诸如大肠杆菌等生物体进行修饰以使其在甲醇上生长的方法，是代谢工程和合成生物学的长期目标。NAD依赖性甲醇脱氢酶（大多数甲醇同化途径中的第一个酶）的动力学性能较差，限制了途径通量，对合成甲基营养障碍提出了严峻挑战。为了解决这个瓶颈，我们使用了甲醛生物传感器来开发噬菌体辅助非连续进化（PANCE）选择甲醇芽孢杆菌甲醇脱氢酶2（Bm Mdh2）的变体。使用此选择，我们开发了Mdh2变体，其Vmax改善了3.5倍。负责增强活性的突变定位到与III型铁依赖性酒精脱氢酶同源的预测活性位点区域，为未来的甲醇脱氢酶工程策略提出了一个新的关键领域。与以前报道的最先进的甲醇脱氢酶相比，进化的Mdh2变体能够将13C-甲醇同化成中心代谢物。这项工作提供了改进的Mdh2变体，并为细菌细胞中的代谢途径建立了实验室进化方法。

更新日期：2019-03-11

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