Phage-Assisted Evolution of Bacillus methanolicus Methanol Dehydrogenase 2

ACS Synthetic Biology
Timothy B RothDavid R Liu

Abstract

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.

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Citations

Jul 14, 2020·Biotechnology and Bioengineering·Sebastian WenkArren Bar-Even
Feb 6, 2020·Biological & Pharmaceutical Bulletin·Javier Pizarro-Bauerle, Hiroki Ando
Feb 12, 2020·Nature Biotechnology·Shannon M MillerDavid R Liu
Feb 12, 2020·Nature Chemical Biology·Seohyoung KimArren Bar-Even
Jul 19, 2020·World Journal of Microbiology & Biotechnology·Philibert Tuyishime, Jean Paul Sinumvayo
Jul 25, 2019·Nature Biotechnology·Benjamin W ThuronyiDavid R Liu
Aug 23, 2019·Current Opinion in Biotechnology·Maciek R Antoniewicz
Feb 5, 2021·Acta Biochimica Et Biophysica Sinica·Huan QiSheng-Ce Tao
Jan 11, 2021·Current Opinion in Biotechnology·Constantinos Katsimpouras, Gregory Stephanopoulos
Nov 18, 2020·Nature Protocols·Shannon M MillerDavid R Liu
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Jun 20, 2020·Biotechnology Advances·Jinyu CuiWeiwen Zhang
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Jun 17, 2021·Biotechnology and Bioengineering·Chunjun ZhanYun Chen
Jul 29, 2021·ACS Synthetic Biology·Jeffrey A DeweyBryan C Dickinson
Oct 15, 2021·Nature Communications·Mary S MorrisonDavid R Liu
Feb 13, 2022·Nucleic Acids Research·Ibrahim S Al'AbriNathan Crook

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Methods Mentioned

BETA
biosensors
fluorescence-activated cell-sorting
FACS
PCR
Assay
Protein Assay

Software Mentioned

SoftMax Pro
Phyre
SeqMan
gnuplot
PANCE

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