Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli

Abstract

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.

Metabolomics for the design of new metabolic engineering strategies for improving aerobic succinic acid production in Escherichia coli

“…This study aim was to use metabolomics tools to identify key metabolites as targets for potential genetic modifications of E. coli strains to improve succinic acid production under aerobic conditions. To this end, three mutant strains, previously described as capable of producing succinic acid under aerobic conditions (Lin et al 2005b ), were constructed: M4, M4- ΔiclR , M4- Δgnd mutant strains (Soto-Varela et al, 2021 ). Comparative analysis of the metabolome using complementary techniques such as FT-IR spectroscopy, GC–MS and targeted HPLC, revealed interesting differences in the behavior and phenotypic profiles of these three mutants.…”

“…The preparation of competent cells, construction of DNA fragments and plasmid transformations were performed using standard procedures (Sambrook et al, 1989 ). The quadruple mutant ΔsdhAΔack-ptaΔpox:: kan (M4) and the quintuple mutant (M4- Δgnd and M4- ΔiclR ) strains were constructed in our laboratory following the homologous recombination method described by Datsenko and Wanner ( 2000 ) as described (Soto-Varela et al, 2021 ). Additionally, the M4- Δgnd and M4- ΔiclR were used as parental strains for the construction of the M4- ΔgndΔmtlD, M4- ΔiclRΔmtlD and M4- ΔiclRΔotsA mutant strains using the same methodology.…”

“…Assays were carried out in batch culture at 37 °C in a rotary shaker at 200 rpm using. The M9 mineral medium composition in 1 L: 9.97 g Na 2 HPO 4 , 3 g KH 2 PO 4 , 1 g NH 4 Cl, 0.5 g NaCl, 2 mL MgSO 4 1 M, 0.1 mL CaCl 2 1 M, 3 mL trace element solution (2.3 g/L FeCl 3 –6H 2 O, 0.039 g/L CuSO 4 –5H 2 O, 0.049 g/L ZnSO 4 –7H 2 O, 0.32 g/L MnCl 2 –7H 2 O, 0.129 g/L CoCl 2 –6H 2 O, 0.037 g/L (NH 3 ) 6 Mo 7 O 24 –4H 2 O and 0.25 g/L H 3 BO 3 ) with 11.5 g/L (125 mM) of glycerol and culture conditions were described in (Soto-Varela et al, 2021 ) with an initial biomass concentration of 0.031 g CDW/L. For metabolomic analysis, the fermentation was performed using M9 medium supplemented with 2 g/L NaHCO 3 for 72 h and samples were taken at 0, 10, 24, 31, 48, 55 and 72 h, in M4, M4- Δgnd and M4- ΔiclR strains.…”

“…For metabolomic analysis, the fermentation was performed using M9 medium supplemented with 2 g/L NaHCO 3 for 72 h and samples were taken at 0, 10, 24, 31, 48, 55 and 72 h, in M4, M4- Δgnd and M4- ΔiclR strains. The fermentation for validation of metabolomic analysis were performed up to 48 h in M4- Δgnd , ΔgndΔmtlD , M4- ΔiclR , M4- ΔiclRΔmtlD and M4- ΔiclRΔotsA strains using M9 medium supplemented with 4 g/L NaHCO 3 after optimization of culture medium by Soto-Varela et al ( 2021 ) for 48 h since inoculation and samples were taken at 0, 10, 24, 31 and 48 h.…”

“…All of the gene knockout strains were verified by PCR using external and Kanamycin internal primers as shown in Table S1. (Soto-Varela et al, 2021) with an initial biomass concentration of 0.031 g CDW/L. For metabolomic analysis, the fermentation was performed using M9 medium supplemented with 2 g/L NaHCO 3 for 72 h and samples were taken at 0, 10, 24, 31, 48, 55 and 72 h, in M4, M4-Δgnd and M4-ΔiclR strains.…”