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

Currently, alpha-ketoglutaric acid (alpha-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 alpha-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 Delta N-LAAD in E.coli BL 21, and then the soluble and active Delta N-LAAD was obtained by the solubilization and renaturation of Delta N-LAAD. Furthermore, the biochemical properties of the refolded Delta N-LAAD were characterized and compared with those of full-length L-AAD. Finally, the Delta N-LAAD was used to synthesize alpha-KG and the maximal formation rate of alpha-KG reached 12.6% (w/w) at 6 h under the following conditions: 12 g/L L-glutamic acid, 0.1 g/L Delta N-LAAD, 5 mM MgCl2, temperature 45 degrees 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 alpha-KG production. (C) 2013 Elsevier B.V. All rights reserved.