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

• Published in: Metabolic engineering Vol. 57; no. C; pp. 247 - 255
• Main Authors: Rohlhill, Julia, Gerald Har, Jie Ren, Antoniewicz, Maciek R., Papoutsakis, Eleftherios T.
• Format: Journal Article
• Language: English
• Published: Belgium Elsevier Inc 01.01.2020; Elsevier
• Subjects: Dynamic regulation; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Formaldehyde - metabolism; Gene Expression Regulation, Bacterial; Glutamic Acid - biosynthesis; Glutamic Acid - genetics; Metabolic Engineering; Methanol; Methanol - metabolism; Microorganisms, Genetically-Modified - genetics; Microorganisms, Genetically-Modified - metabolism; Pentose phosphate pathway; Pentose Phosphate Pathway - genetics; Redox balance; Synthetic methylotrophy; Synthetic methylotrophy; Pentose phosphate pathway; Redox balance; Dynamic regulation; Escherichia coli; Methanol
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2019.12.006

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 Pfrm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under Pfrm 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. •Expression of PPP genes rpe and tkt under dynamic formaldehyde control improves methanol carbon assimilation.•Deletion of NAD-dependent malate dehydrogenase improves biomass generation on methanol and yeast extract co-substrate.•Short-term 13C-methanol assay demonstrates expression of methanol consumption enzymes in Δmaldh strain with rpe and tkt.•Achieved high carbon and energy utilization from methanol through the Pfrm expression of rpe and tkt in the Δmaldh strain.