l-Theanine production in a fed-batch culture.

Cultures of recombinant E. coli W3110S3GK were incubated in 250 ml of seed medium (10 g/liter of peptone, 5 g/liter of yeast extract, 5 g/liter of NaCl, 15 g/liter of glucose, and 3 g/liter of CaCO₃) containing 100 mg/liter of ampicillin in a 1-liter Erlenmeyer flask at 30°C for 16 h with shaking (220 rpm). The culture (37.5 ml) was transferred into a 3-liter jar fermentor (Mitsuwa Frontech, Osaka, Japan) containing 750 ml of main medium [20 g/liter of glucose, 6 g/liter of Na₂HPO₄, 3 g/liter of KH₂PO₄, 5 g/liter of NaCl, 3 g/liter of (NH₄)₂SO₄, 5 g/liter of yeast extract, 10 mg/liter of MnSO₄·7H₂O, 2 g/liter of MgSO₄·H₂O, 200 mg/liter of FeSO₄·7H₂O, 10 mg/liter of thiamine hydrochloride, and 0.2 ml of defoamer LG109; Adeka, Tokyo, Japan] with 200 mg/liter of carbenicillin and cultivated at 30°C with aeration (0.75 liter/min) and agitation (800 rpm). After 8 h, IPTG (1.0 mM final concentration) was added to induce the expression of recombinant proteins. When the initial glucose was depleted, continuous feeding of a glucose solution (500 g/liter) was begun. Glucose solution was supplied at a controlled rate (10 ml/h for TEA4 Δggt and 4 ml/h for TEA6 Δggt) so as not to exceed 50 g/liter of glucose in the main culture. During cultivation, the pH was maintained at 7.0 with 14% (vol/vol) NH₄OH. At each time point (Fig. 7), a sample (5 ml) was taken, and a portion (1 ml) was used to measure optical density at 660 nm. The remainder of the sample was immediately centrifuged, and the supernatant was used to determine the concentrations of glucose, amino acids, and l-theanine.