Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation

Julia Rohlhill; Jie Ren Gerald Har; Maciek R Antoniewicz; Eleftherios T Papoutsakis

doi:10.1016/j.ymben.2019.12.006

Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation

Metab Eng. 2020 Jan:57:247-255. doi: 10.1016/j.ymben.2019.12.006. Epub 2019 Dec 24.

Authors

Julia Rohlhill¹, Jie Ren Gerald Har², Maciek R Antoniewicz², Eleftherios T Papoutsakis³

Affiliations

¹ Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St, Newark, DE, 19716, USA; Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE, 19711, USA.
² Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St, Newark, DE, 19716, USA.
³ Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy St, Newark, DE, 19716, USA; Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way, Newark, DE, 19711, USA. Electronic address: epaps@udel.edu.

PMID: 31881281
DOI: 10.1016/j.ymben.2019.12.006

Abstract

Escherichia coli is an ideal choice for constructing synthetic methylotrophs capable of utilizing the non-native substrate methanol as a carbon and energy source. All current E. coli-based synthetic methylotrophs require co-substrates. They display variable levels of methanol-carbon incorporation due to a lack of native regulatory control of biosynthetic pathways, as E. coli does not recognize methanol as a proper substrate despite its ability to catabolize it. Here, using the E. coli formaldehyde-inducible promoter P_frm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under P_frm control exhibited 8- to 30-fold transcriptional upregulation during growth on methanol. Formaldehyde-induced episomal expression of the B. methanolicus rpe and tkt genes involved in the regeneration of ribulose 5-phosphate required for formaldehyde fixation led to significantly improved methanol assimilation into intracellular metabolites, including a 2-fold increase of ¹³C-methanol into glutamate. Using a simple strategy for redox perturbation by deleting the E. coli NAD-dependent malate dehydrogenase gene maldh, we demonstrate 5-fold improved biomass formation of cells growing on methanol in the presence of a small concentration of yeast extract. Further improvements in methanol utilization are achieved via adaptive laboratory evolution and heterologous rpe and tkt expression. A short-term in vivo¹³C-methanol labeling assay was used to determine methanol assimilation activity for Δmaldh strains, and demonstrated dramatically higher labeling in intracellular metabolites, including a 6-fold and 1.8-fold increase in glycine labeling for the rpe/tkt and evolved strains, respectively. The combination of formaldehyde-controlled pentose phosphate pathway expression and redox perturbation with the maldh knock-out greatly improved both growth benefit with methanol and methanol carbon incorporation into intracellular metabolites.

Keywords: Dynamic regulation; Escherichia coli; Methanol; Pentose phosphate pathway; Redox balance; Synthetic methylotrophy.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Escherichia coli* / genetics
Escherichia coli* / metabolism
Formaldehyde / metabolism*
Gene Expression Regulation, Bacterial*
Glutamic Acid / biosynthesis
Glutamic Acid / genetics
Metabolic Engineering*
Methanol / metabolism
Microorganisms, Genetically-Modified* / genetics
Microorganisms, Genetically-Modified* / metabolism
Pentose Phosphate Pathway / genetics*

Substances

Formaldehyde
Glutamic Acid
Methanol