Construction of a Synthetic Bypass for Improvement of Aerobic Synthesis of Succinic Acid through the Oxidative Branch of the Tricarboxylic Acid Cycle by Recombinant Escherichia coli Strains

Using Escherichia coli MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆fadE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, PL-SDφ10-tesB, ∆yciA as a core strain, derivatives capable of synthesizing adipic acid from glucose through the inverted fatty acid β-oxidation pathway were obtained. Biosynthesis of the target compound by recombinants was ensured by the primary condensation of acetyl-CoA and succinyl-CoA by 3-oxoacyl-CoA thiolase PaaJ and the catalysis of the final reaction of the cycle by acyl-CoA dehydrogenases FadE and FabI. Deletion in the strains of sucCD genes encoding components of succinyl-CoA synthase did not increase the relative intracellular availability of succinyl-CoA for target biosynthetic reactions and did not lead to an increase in adipic acid accumulation by the recombinants. The secretion of succinic and malic acids by the strains with an impaired tricarboxylic acid cycle remained almost unchanged, indicating the activity in the cells of glyoxylate shunt reactions that compete with the cycle reactions for isocitrate, required for succinyl-CoA formation. When isocitrate lyase, malate synthases A and G, and bifunctional kinase/phosphatase of isocitrate dehydrogenase were inactivated in strains due to deletion of the aceBAK operon genes and glcB, adipic acid synthesis by recombinants increased three-fold and reached 0.33 mM.

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The Effect of Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid through Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

Gulevich

Skorokhodova

Дебабов

2023

Appl Biochem Microbiol

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Optimization of Aerobic Synthesis of Succinic Acid from Glucose by Recombinant Escherichia coli Strains through the Tricarboxylic Acid Cycle Variant Mediated by the Action of 2-Ketoglutarate Decarboxylase

Skorokhodova,

Gulevich,

Debabov

2023

Appl Biochem Microbiol

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Engineering Escherichia coli for Efficient Aerobic Conversion of Glucose to Malic Acid through the Modified Oxidative TCA Cycle

Skorokhodova,

Stasenko,

Krasilnikova

et al. 2022

Fermentation

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Malic acid is a versatile building-block chemical that can serve as a precursor of numerous valuable products, including food additives, pharmaceuticals, and biodegradable plastics. Despite the present petrochemical synthesis, malic acid, being an intermediate of the TCA cycle of a variety of living organisms, can also be produced from renewable carbon sources using wild-type and engineered microbial strains. In the current study, Escherichia coli was engineered for efficient aerobic conversion of glucose to malic acid through the modified oxidative TCA cycle resembling that of myco- and cyanobacteria and implying channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation. The formation of succinate semialdehyde was enabled in the core strain MAL 0 (∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PL-glk, Ptac-galP, ∆aceBAK, ∆glcB) by the expression of Mycobacterium tuberculosis kgd gene. The secretion of malic acid by the strain was ensured, resulting from the deletion of the mdh, maeA, maeB, and mqo genes. The Bacillus subtilis pycA gene was expressed in the strain to allow pyruvate to oxaloacetate conversion. The corresponding recombinant was able to synthesise malic acid from glucose aerobically with a yield of 0.65 mol/mol. The yield was improved by the derepression in the strain of the electron transfer chain and succinate dehydrogenase due to the enforcement of ATP hydrolysis and reached 0.94 mol/mol, amounting to 94% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient strains and processes of bio-based malic acid production.

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Construction of a Synthetic Bypass for Improvement of Aerobic Synthesis of Succinic Acid through the Oxidative Branch of the Tricarboxylic Acid Cycle by Recombinant Escherichia coli Strains

Cited by 3 publications

References 28 publications

The Effect of Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid through Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

The Effect of Glyoxylate Shunt Inactivation on Biosynthesis of Adipic Acid through Inverted Fatty Acid β-Oxidation by Escherichia coli Strains

Optimization of Aerobic Synthesis of Succinic Acid from Glucose by Recombinant Escherichia coli Strains through the Tricarboxylic Acid Cycle Variant Mediated by the Action of 2-Ketoglutarate Decarboxylase

Engineering Escherichia coli for Efficient Aerobic Conversion of Glucose to Malic Acid through the Modified Oxidative TCA Cycle

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