Increased 3-hydroxypropionic acid production from glycerol, by modification of central metabolism in Escherichia coli

Abstract

Background3-hydroxypropionic acid (3HP) is an important chemical precursor for the production of bioplastics. Microbial production of 3HP from glycerol has previously been developed through the optimization of culture conditions and the 3HP biosynthesis pathway. In this study, a novel strategy for improving 3HP production in Escherichia coli was investigated by the modification of central metabolism based on a genome-scale metabolic model and experimental validation.ResultsMetabolic simulation identified the double knockout of tpiA and zwf as a candidate for improving 3HP production. A 3HP-producing strain was constructed by the expression of glycerol dehydratase and aldehyde dehydrogenase. The double knockout of tpiA and zwf increased the percentage carbon-molar yield (C-mol%) of 3HP on consumed glycerol 4.4-fold (20.1 ± 9.2 C-mol%), compared to the parental strain. Increased extracellular methylglyoxal concentrations in the ΔtpiA Δzwf strain indicated that glycerol catabolism was occurring through the methylglyoxal pathway, which converts dihydroxyacetone phosphate to pyruvate, as predicted by the metabolic model. Since the ΔtpiA Δzwf strain produced abundant 1,3-propanediol as a major byproduct (37.7 ± 13.2 C-mol%), yqhD, which encodes an enzyme involved in the production of 1,3-propanediol, was disrupted in the ΔtpiA Δzwf strain. The 3HP yield of the ΔtpiA Δzwf ΔyqhD strain (33.9 ± 1.2 C-mol%) was increased 1.7-fold further compared to the ΔtpiA Δzwf strain and by 7.4-fold compared to the parental strain.ConclusionThis study successfully increased 3HP production by 7.4-fold in the ΔtpiA Δzwf ΔyqhD E. coli strain by the modification of the central metabolism, based on metabolic simulation and experimental validation of engineered strains.

Eliminating acetate formation improves citramalate production by metabolically engineered Escherichia coli

“…For instance, zwf gene deletion enhanced lycopene production by over 130% in recombinant E. coli strains, owing to an improved Emden–Meyerhof–Parnas (EMP) pathway flux and increased pyruvate [44]. Similarly, a 4.4-fold increase in yield was reported in an E. coli tpiA zwf strain generating 3-hydroxypropionic acid from glycerol [35]. In our shake flask studies, strains with zwf deletions resulted in much lower growth and citramalate accumulation.…”

Dual synthetic pathway for 3-hydroxypropionic acid production in engineered Escherichia coli

“…There are reports of improvement of 3-HP production by deletion of the yqhD gene in E. coli with ALDH pathway (19,24). Based on those reports, we expect that deletion of yqhD from our strains will provide further elevation of 3-HP production.…”

Designer Microorganisms for Optimized Redox Cascade Reactions – Challenges and Future Perspectives

“…This was achieved with ag ene KO simulation prior to constructing the strain in vivo. [168] Apart from FBA, the impact of cascades which are coupled to the carbon metabolism of am icroorganism can be evaluated via 13 Cm etabolicf lux analysis.T his method is usedt oa nalyze the flux distributionso ft he centralm etabolism.T herefore,e xperiments with [1][2][3][4][5][6][7][8][9][10][11][12][13] C] andu niformlyc arbon [U- 13 C]-labelledg lucose as only carbon source are performed. Cultures are grown on labelled substrates andh arvested during steady-state.P roteins are hydrolyzed and amino acids are derivatized for GC-MS measurements.…”