13c-Metabolic Flux Analysis for Mevalonate-Producing Strain of Escherichia coli

Mevalonate (MVA) is used to produce various useful products such as drugs, cosmetics and food additives, therefore the biological production of MVA is highly desired. An MVA-producing strain of Escherichia coli was constructed by introducing mvaES genes from Enterococcys faecalis. These enzymes catalyze the condensation of acetyl-CoA and reduction of them using NADPH. The distribution of flux at acetyl-CoA node and the flux of NADPH generation to drive the MVA biosynthesis pathway may effect on the MVA productivity. In the present study, the effect of MVA production on the central carbon metabolism was investigated by ¹³C-metabolic flux analysis. This analysis is a method used to estimate the intracellular reaction rate of pathways based on ¹³C-labelling experiment.

Materials and methods

The control and engineered strains were cultured in synthetic medium with [1-¹³C] glucose as sole carbon source. The ¹³C-experiment of proteinogenic amino acids at the exponential growth phase was measured by GC-MS. Metabolic fluxes were estimated by minimizing the residual sum of squares between the experimentally measured and model predicted ¹³C-enrichment. The 95% confidence intervals of each flux were calculated by a grid search method. These flux estimations were performed by in-house software OpenMebius. The relationship between MVA production rate and each flux variability (flux solution space) was calculated on Matlab using CobraToolbox.

Results and discussion

The engineered strain produced 1.84 mmol/gDCW/h yielding 22% (C-mol/C-mol) of MVA from glucose in the aerobic exponential growth phase. Acetate production rate in the engineered strain was ten times lower than the control strain and MVA was produced by only the engineered strain. The response to MVA production in central metabolic pathway was discussed the differences of flux distribution between control and engineered strains. Although 45% and 43% of acetyl-CoA was diverted to acetate production and to the tricarboxylic acid cycle in the control strain, whereas the ratios were decreased to 4% and 27% in the engineered strain. It seems that acetyl-CoA was diverted to MVA synthesis pathway in the engineered strain due to overexpression of enzymes of the pathway. NADPH production is important for MVA synthesis as it provides the reducing power for MVA synthesis. Although the oxidative pentose-phosphate pathway is considered as the NADPH generating pathway in E. coli, the flux was not significantly different between the control and engineered strains. The production/consumption balance of NADPH suggested that most NADPH for MVA synthesis was obtained from the transhydrogenase reaction in the engineered strain. The flux solution space predicted that repression of citrate synthase is effective for enhancing MVA production.