Effect of the Tricarboxylic Acid Cycle Intensification on biosynthesis of Adipic Acid Through the Inverted Fatty Acid β-oxidation by Escherichia coli Strains

Using previously engineered adipate-secreting Escherichia MG1655 lacIQ, ∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, PL-SDφ10-atoB, Ptrc-ideal-4-SDφ10-fadB, ∆fadE, PL-SDφ10-tesB, ∆yciA, Ptrc-ideal-4-SDφ10-fabI, PL-SDφ10-paaJ, ∆aceBAK, ∆glcB as a core strain, the derivatives capable of enhanced synthesis of the target compound from glucose via the reversed fatty acid β-oxidation pathway were obtained. The respective effect was achieved due to the intensification of the tricarboxylic acid cycle in the cells. Prevention of multiple cycle turnovers, resulting from the inactivation of succinate dehydrogenase, had no pronounced effect on the formation of adipic acid by the recombinant. Upon the cycle intensification due to enhancing anaplerotic oxaloacetic acetic acid formation from phosphoenolpyruvate, resulting from the increased expression of the native ppc gene, the synthesis of adipic acid rose 1.2-fold to ~390 μM. Enabling the formation of oxaloacetate from pyruvic acid, by introducing in the cells of heterologous Bacillus subtilis pyruvate carboxylase, resulted in a 1.5-fold intensification of the cycle, concomitantly with the proportional increase in adipic acid secretion to ~496 μM. Subsequent inactivation of sdhAB genes in the strain increased the secretion of the target compound only slightly and adipic acid titer reached ~520 μM. The obtained data indicated a direct dependence of the efficiency of adipic acid synthesis by the engineered strains on the degree of intensification of the tricarboxylic acid cycle.