| Summary: | 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, <i>Escherichia coli</i> 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 (∆<i>ackA-pta</i>, ∆<i>poxB</i>, ∆<i>ldhA</i>, ∆<i>adhE</i>, ∆<i>ptsG</i>, P<sub>L</sub>-<i>glk</i>, P<i><sub>tac</sub></i>-<i>galP</i>, ∆<i>aceBAK</i>, ∆<i>glcB</i>) by the expression of <i>Mycobacterium tuberculosis kgd</i> gene. The secretion of malic acid by the strain was ensured, resulting from the deletion of the <i>mdh</i>, <i>maeA</i>, <i>maeB</i>, and <i>mqo</i> genes. The <i>Bacillus subtilis pycA</i> 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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