One-step production of α-ketoglutaric acid from glutamic acid with an engineered l-amino acid deaminase from Proteus mirabilis

Currently, α-ketoglutaric acid (α-KG) is industrially produced by multi-step chemical synthesis, which can cause heavy environmental pollution. Here we reported a simple one-step approach for the production of α-KG by transforming l-glutamic acid with an engineered l-amino acid deaminase (l-AAD) from Proteus mirabilis. First, to facilitate the purification of membrane-bound l-AAD, one N-terminal transmembrane region (from 21 to 87th nucleotide) was removed from l-AAD to block the binding of l-AAD with membrane, and the relatively low-usage codons were replaced by high-usage codons in Escherichia coli to improve the expression level. However, inclusion bodies formed when expressing the ΔN-LAAD in E. coli BL 21, and then the soluble and active ΔN-LAAD was obtained by the solubilization and renaturation of ΔN-LAAD. Furthermore, the biochemical properties of the refolded ΔN-LAAD were characterized and compared with those of full-length l-AAD. Finally, the ΔN-LAAD was used to synthesize α-KG and the maximal formation rate of α-KG reached 12.6% (w/w) at 6 h under the following conditions: 12 g/L l-glutamic acid, 0.1 g/L ΔN-LAAD, 5 mM MgCl2, temperature 45 °C and pH 8.0. Compared with the multi-step chemical synthesis, the transformation approach has less environmental pollution and has a great potential for α-KG production.

► This is the first systematic report about the a-KG production by biotransformation approach.
► The l-AAD from Proteus mirabilis was engineered to make it not membrane-bound.
► The active l-AAD was obtained by the solubilization and refolding of l-AAD inclusion bodies.
► The highest a-KG yield reached 12.6% using 12.0 g/L glutamic acid, 0.1 g/L enzyme, 5 mM MgCl2.
► Mg2+ had stimulation effects, while glutamic acid had competitive inhibitions on transformation.