Metabolic engineering of Escherichia coli for biosynthesis of inosinic acid

Abstract

Microbial fermentation is one of the primary approaches for inosinic acid (IMP) production. However, most IMP-producing strains are non-model organisms, which limits the application of genetic engineering for further strain improvement. Additionally, these strains require expensive substrates as feedstock, increasing production costs. In this study, we engineered Escherichia coli, a well-characterized model microorganism, as the chassis for IMP biosynthesis from glucose by systematically optimizing its metabolic network. First, we reprogrammed the metabolic flux of the pentose phosphate pathway to increase the intracellular availability of phosphoribosyl pyrophosphate (PRPP), a key precursor of IMP, achieving an IMP titer of 484.6 mg/L. Then, the alleviation of feedback inhibition in the purine biosynthetic pathway increased the IMP titer to 562.0 mg/L. Furthermore, by identifying the rate-limiting steps in the purine synthesis pathway and knocking out competing pathways for IMP synthesis, we further increased the IMP titer to 1409.6 mg/L. Finally, by enhancing the supply of cofactor N₁₀-formyl-tetrahydrofolate, the titer of IMP reached 2.1 g/L in shake-flasks and 3.1 g/L in 5-L bioreactors. This study provides new insights for the construction of cell factories for the synthesis of nucleotide derivatives.

Keywords

Inosinic acid / Escherichia coli / Metabolic engineering / N₁₀-formyl-tetrahydrofolate

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Funding

the National Key Research and Development Program of China(2022YFC3401303)

the Key Technological Project of Jiangxi Province(20244AFH82001)

the Key R&D Program of Shandong Province, China(2024CXGC010917)

RIGHTS & PERMISSIONS

Jiangnan University