Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

R Kyle Bennett¹, Michael Dillon², Jie Ren Gerald Har³, Alec Agee⁴, Bryan von Hagel⁵, Julia Rohlhill⁶, Maciek R Antoniewicz⁷, Eleftherios T Papoutsakis⁸

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: rkbenne@udel.edu.
² Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: miked@udel.edu.
³ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA. Electronic address: harjrg@udel.edu.
⁴ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: aagee@udel.edu.
⁵ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: bryanvh@udel.edu.
⁶ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: juliapet@udel.edu.
⁷ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA. Electronic address: mranton@udel.edu.
⁸ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: epaps@udel.edu.

PMID: 32179162
DOI: 10.1016/j.ymben.2020.03.003

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

R Kyle Bennett et al. Metab Eng. 2020 Jul.

. 2020 Jul:60:45-55.

doi: 10.1016/j.ymben.2020.03.003. Epub 2020 Mar 13.

Authors

R Kyle Bennett¹, Michael Dillon², Jie Ren Gerald Har³, Alec Agee⁴, Bryan von Hagel⁵, Julia Rohlhill⁶, Maciek R Antoniewicz⁷, Eleftherios T Papoutsakis⁸

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: rkbenne@udel.edu.
² Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: miked@udel.edu.
³ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA. Electronic address: harjrg@udel.edu.
⁴ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: aagee@udel.edu.
⁵ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: bryanvh@udel.edu.
⁶ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: juliapet@udel.edu.
⁷ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA. Electronic address: mranton@udel.edu.
⁸ Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE, USA. Electronic address: epaps@udel.edu.

PMID: 32179162
DOI: 10.1016/j.ymben.2020.03.003

Abstract

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h^-1 in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.

Keywords: Escherichia coli; Glucose; Methanol; Methanol-dependence; Synthetic methylotrophy.

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Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

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Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

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