Functional evaluation of non-oxidative glycolysis in Escherichia coli in the stationary phase under microaerobic conditions

• Published in: Journal of bioscience and bioengineering Vol. 135; no. 4; pp. 291 - 297
• Main Authors: Miyoshi, Kenta, Kawai, Ryutaro, Niide, Teppei, Toya, Yoshihiro, Shimizu, Hiroshi
• Format: Journal Article
• Language: English
• Published: Japan Elsevier B.V 01.04.2023
• Subjects: Acetates - metabolism; Acetyl Coenzyme A - metabolism; Acetyl-CoA; Carbon - metabolism; Carbon Dioxide - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Flux balance analysis; Flux estimation; Flux variability analysis; Glucose - metabolism; Glycolysis; Metabolic Engineering; Non-oxidative glycolysis; Stationary phase; Flux balance analysis; Escherichia coli; Flux estimation; Acetyl-CoA; Flux variability analysis; Non-oxidative glycolysis; Stationary phase
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2023.01.002

In microbial bioproduction, CO2 emissions via pyruvate dehydrogenase in the Embden-Meyerhof pathway, which converts glucose to acetyl-CoA, is one of the challenges for enhancing carbon yield. The synthetic non-oxidative glycolysis (NOG) pathway transforms glucose into three acetyl-CoA molecules without CO2 emission, making it an attractive module for metabolic engineering. Because the NOG pathway generates no ATP and NADH, it is expected to use a resting cell reaction. Therefore, it is important to characterize the feasibility of the NOG pathway during stationary phase. Here, we experimentally evaluated the in vivo metabolic flow of the NOG pathway in Escherichia coli. An engineered strain was constructed by introducing phosphoketolase from Bifidobacterium adolescentis into E. coli and by deleting competitive reactions. When the strain was cultured in magnesium-starved medium under microaerobic conditions, the carbon yield of acetate, an end-product of the NOG pathway, was six times higher than that of the control strain harboring an empty vector. Based on the mass balance constraints, the NOG flux was estimated to be between 2.89 and 4.64 mmol g−1 h−1, suggesting that the engineered cells can convert glucose through the NOG pathway with enough activity for bioconversion. Furthermore, to expand the application potential of NOG pathway-implemented strains, the theoretical maximum yields of various useful compounds were calculated using flux balance analysis. This suggests that the theoretical maximum yields of not only acetate but also lactam compounds can be increased by introducing the NOG pathway. This information will help in future applications of the NOG pathway. [Display omitted] •Non-oxidative glycolysis (NOG) converts glucose to acetyl-CoA without CO2 emissions.•Engineered Escherichia coli strains with introducing phosphoketolase were constructed.•Cultural profiles in stationary phase under microaerobic conditions were evaluated.•The NOG flux was experimentally evaluated by flux variability analysis.•Theoretical candidates for achieving a higher carbon yield using NOG were predicted.