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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.<\/jats:p>","DOI":"10.1021\/acssynbio.8b00481","type":"journal-article","created":{"date-parts":[[2019,3,11]],"date-time":"2019-03-11T17:33:00Z","timestamp":1552325580000},"page":"796-806","source":"Crossref","is-referenced-by-count":107,"title":["Phage-Assisted Evolution of\n                    <i>Bacillus methanolicus<\/i>\n                    Methanol Dehydrogenase 2"],"prefix":"10.1021","volume":"8","author":[{"given":"Timothy B.","family":"Roth","sequence":"first","affiliation":[{"name":"Broad Institute of MIT and Harvard , , , ,","place":["Cambridge, Massachusetts, United States, 02142"]},{"name":"Harvard University , , , ,","place":["Cambridge, Massachusetts, United States, 02138"]},{"name":"Harvard University , , , ,","place":["Cambridge, Massachusetts, United States, 02138"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin M.","family":"Woolston","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6909-4568","authenticated-orcid":true,"given":"Gregory","family":"Stephanopoulos","sequence":"additional","affiliation":[{"name":"Massachusetts Institute of Technology , , , ,","place":["Cambridge, Massachusetts, United States, 02139"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9943-7557","authenticated-orcid":true,"given":"David R.","family":"Liu","sequence":"additional","affiliation":[{"name":"Broad Institute of MIT and Harvard , , , ,","place":["Cambridge, Massachusetts, United States, 02142"]},{"name":"Harvard University , , , ,","place":["Cambridge, Massachusetts, United States, 02138"]},{"name":"Harvard University , , , ,","place":["Cambridge, Massachusetts, United States, 02138"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2019,3,11]]},"reference":[{"key":"2026090223414956500_cit1","doi-asserted-by":"publisher","first-page":"5352","DOI":"10.1038\/ncomms6352","article-title":"A system for the\ncontinuous directed evolution of proteases rapidly reveals drug-resistance\nmutations","volume":"5","author":"Dickinson","year":"2014","journal-title":"Nat. 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