[Efficient synthesis of L-theanine by recombinant strain Corynebacterium glutamicum SYPA5-5]

To construct a Corynebacterium glutamicum strain system for L-theanine production by the secretion expression of γ-glutamyltranspeptidase.

Two genes ggt and △sp ggt (without signal peptide fragment) from Bacillus subtilis were cloned and expressed in C. glutamicum. Then the recombinant GGT was used for L-theanine production under the optimal conditions: GGT enzyme 0.9 U/mL, pH 10, 37℃, and 20 mmol/L L-glutamine and 60 mmol/L ethylamine were fed every two hours. Supplementation was ceased after 12 h to minimize the substrates residue in the final broth.

Firstly, two different recombinant C. glutamicum strains C. glutamicum SYPA5-5/pXMJ19-△sp ggt (without signal peptide), C. glutamicum SYPA5-5/pXMJ19-ggt (with signal peptide) were successfully constructed. In comparison to C. glutamicum SYPA5-5/pXMJ19-△sp ggt, C. glutamicum SYPA5-5/pXMJ19-ggt showed the ability to secret GGT into the medium and 5-fold higher enzyme activity than that in the former strain. This finding suggested that the signal peptide of GGT was responsible for the secretion and could work in C.glutamicum strain system. Furthermore, the glucose concentration and the adding time of inducer IPTG on GGT production were optimized in shake-flask. The batch transformation conditions were also investigated. The optimal ratio of L-glutamine to ethylamine was 1:3, and optimal enzyme amount was 0.06 U/mL. The highest L-theanine production reached at 104.36 mmol/L at 12 h with the conversion rate of 86.9%.

This is the first time to use the C. glutamicum system for the efficient synthesis of L-theanine. Furthermore, the signal peptide of GGT is identified to function well in C. glutamicum, providing a possible strategy for constructing a secretion expression system in C. glutamicum.