期刊
ACS SYNTHETIC BIOLOGY
卷 8, 期 4, 页码 796-806出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00481
关键词
methanol dehydrogenase; synthetic methylotrophy; methanol assimilation; directed evolution; phage-assisted continuous evolution; phage-assisted noncontinuous evolution
资金
- US NIH NIBIB [RO1 EB022376]
- NIGMS [R35 GM118062]
- US DOE [DE-AR0000433]
- Howard Hughes Medical Institute
- NSF GRFP [1144152, 1122374]
- Direct For Education and Human Resources
- Division Of Graduate Education [1144152] Funding Source: National Science Foundation
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 V-max. 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 C-13-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.
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