PPK和GMAS共表达重组菌株的构建及其在L-茶氨酸合成中的应用

Construction of recombinant strains co-expressing PPK and GMAS for the synthesis of L-theanine

摘要:

构建了共表达ATP再生和L-茶氨酸合成酶的重组大肠杆菌菌株，并将其应用于L-茶氨酸的合成中。合成磷酸激酶 (PPK) 和γ-谷氨酰甲胺合成酶 (GMAS) 基因序列，分别利用pETDuet-1和pET-21a(+) 载体，构建共表达重组质粒pETDuet-ppk+gmas和pET21a-ppk+gmas。将上述两种重组质粒转入大肠杆菌BL21(DE3) 中，获得重组菌株TPG和APG。IPTG诱导表达后，SDS-PAGE结果表明，PPK和GMAS在两种重组菌中均可溶性表达。当用于催化L-茶氨酸合成时，来自APG的GMAS-PPK要优于TPG。利用APG所产的酶进行L-茶氨酸合成，在37 ℃、pH 7.0条件下，使用催化量ATP可实现L-茶氨酸的摩尔产率为86.0%。该结果一方面扩展了酶法ATP再生系统的应用，另一方面为生物催化合成L-茶氨酸提供了一种有效方法。

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.