Sequential flux optimization to improve GABA synthesis from acetate in Escherichia coli

Abstract

Strategic allocation of metabolic flux is essential for achieving higher production performance in genetically engineered organisms. Flux optimization between cell growth and chemical production has led to the establishment of cost-effective chemical production methods in microbial cell factories. This effect is amplified when utilizing a low-cost carbon source. γ-aminobutyric acid (GABA) is an important component in pharmaceutical industry and for the production of biodegradable polymers. Acetate is an attractive and cost-effective substrate for the biosynthetic production of value-added chemicals, including GABA. However, a balanced distribution of acetate-derived flux is essential for optimizing production performance without compromising growth. In this study, we demonstrated GABA production from acetate by focusing on optimizing the metabolic flux at critical nodes.

To construct a E. coli W strain capable of producing and accumulating GABA by utilizing acetate as a sole carbon source, three genes were deleted (iclR, transcriptional regulator of aceBAK genes which is essential for acetate metabolism, gabT and puuE genes, 4-aminobutyrate aminotransferase, which turns GABA into succinate-semialdehyde) and a mutated gadB gene (gadB E89Q Δ452-466) that shows activity at around pH 7 was expressed by cloning it into a pETDuet-1 vector. The resulting strain was named WGM strain, After the initial production of GABA from acetate in the WGM strain, flux rewiring of the sequential metabolite nodes led to a considerable improvement in GABA production. Initial upregulation of the aceA gene in the glyoxylate shunt at the isocitrate node and subsequent downregulation of the sucA gene in the tricarboxylic acid cycle at the α-ketoglutarate node amplified metabolic flux directed to GABA. The resulting strain produced 2.54 g/L GABA from 5 g/L acetate in 24 h, with 0.34 g/g yield.

Through stepwise flux optimization at key metabolic nodes, engineered E. coli W produced GABA from acetate with high efficiency. These findings suggest that delicate flux balancing using an economical substrate can contribute to the cost-effective production of GABA.