Enhancement of alpha-ketoglutaric acid production from l-glutamic acid by high-cell-density cultivation

• Three steps were performed in high-cell-density cultivation of a recombinant E.coli.
• A novel two-stage feeding strategy (exponential feeding and DO-stat) was proposed.
• High l-glutamate oxidase expression level (156.1 U/mL) was obtained.
• High production (127.2 g/L) of α-KG was obtained in 24 h by whole-cell bioconversion.

In this study, an efficient strategy of high-cell-density cultivation was exploited to obtain recombinant l-glutamate oxidase (LGOX), which was used to produce alpha-ketoglutaric acid (α-KG) from l-glutamic acid. First, lactose was used to replace isopropyl β-D-1-thiogalactopyranoside as a cheaper and more effective inducer. Second, a novel, two-stage feeding strategy was proposed, in which the exponential feeding mode was first performed in a 5-L fermenter until the maximum dissolved oxygen (DO) concentration was reached, which was followed by a DO-stat feeding mode. Based on the two-stage feeding strategy and by optimizing the induction strategies, the maximal cell density and LGOX activity reached 48.4 g/L and 156.1 U/mL, respectively, after 20 h of cultivation. When a whole-cell biocatalyst was used, the titer of α-KG was up to 127.2 g/L from 132 g/L l-glutamic acid in 24 h.

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