Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.
Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E. coli were limited by methanol dehydrogenases (Mdhs) with unfavorable enzyme kinetics. We engineered E. coli to utilize methanol using a superior NAD-dependent Mdh from Bacillus stearothermophilus and ribulose monophosphate (RuMP) pathway enzymes from B. methanolicus. Using13C-labeling, we demonstrate this E. coli strain converts methanol into biomass components. For example, the key TCA cycle intermediates, succinate and malate, exhibit labeling up to 39%, while the lower glycolytic intermediate, 3-phosphoglycerate, up to 53%. Multiple carbons are labeled for each compound, demonstrating a cycling RuMP pathway for methanol assimilation to support growth. By incorporating the pathway to synthesize the flavanone naringenin, we demonstrate the first example of in vivo conversion of methanol into a specialty chemical in E. coli.
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA; The Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711, USA. Electronic address: wbwhit@udel.edu.
W Brian
WB
Whitaker
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA; Department of Chemistry, Hamilton College, 198 College Hill Rd., Clinton, NY 13323, USA. Electronic address: jajones@hamilton.edu.
J Andrew
JA
Jones
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA; The Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711, USA. Electronic address: rkbenne@udel.edu.
R Kyle
RK
Bennett
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA. Electronic address: jgonz@udel.edu.
Jacqueline E
JE
Gonzalez
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA. Electronic address: vernav@rpi.edu.
Victoria R
VR
Vernacchio
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA. Electronic address: collis6@rpi.edu.
Shannon M
SM
Collins
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA. Electronic address: mpalmer@udel.edu.
Michael A
MA
Palmer
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA. Electronic address: schm@udel.edu.
Samuel
S
Schmidt
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA. Electronic address: mranton@udel.edu.
Maciek R
MR
Antoniewicz
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA; Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St., Troy, NY 12180, USA. Electronic address: koffam@rpi.edu.
Mattheos A
MA
Koffas
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St., Newark, DE 19716, USA; The Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE 19711, USA. Electronic address: papoutsakis@dbi.udel.edu.
Eleftherios T
ET
Papoutsakis
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000502:physiology; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D044950:Flavanones / Q000096:biosynthesis* / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology*; D000432:Methanol / Q000378:metabolism*
D029968:Escherichia coli Proteins; D044950:Flavanones; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); C005273:naringenin; D000432:Methanol
E. coli; Methanol; Methylotrophy; Pathway engineering for substrate utilization