Construction of recombinant strains co-expressing PPK and GMAS for the synthesis of L-theanine.
- Author:
Yuan LI
1
;
Shan LIU
1
;
Jun ZHU
1
Author Information
- Publication Type:Journal Article
- Keywords: ATP regeneration; L-theanine; co-expression; dual-enzyme coupled reaction; polyphosphate kinase; γ-glutamylmethylamide synthetase
- MeSH: Carbon-Nitrogen Ligases; genetics; Electrophoresis, Polyacrylamide Gel; Escherichia coli; genetics; Glutamates; biosynthesis; Ligases; Phosphotransferases (Phosphate Group Acceptor); genetics
- From: Chinese Journal of Biotechnology 2016;32(12):1745-1749
- CountryChina
- Language:Chinese
- Abstract: Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.
