利用重组钝齿棒杆菌高效合成L-茶氨酸

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

摘要:

[目的]构建Bacillus subtilis来源的γ-谷氨酰转肽酶蛋白（GGT）的Corynebacterium glutamicum SYPA5-5表达系统，验证该蛋白信号肽片段在宿主表达体系中的作用，并将该体系应用于高效合成茶氨酸的研究。[方法]将该ggt基因和切除信号肽的片段基因（Δsp ggt）在C.glutamicum SYPA5-5中克隆表达。以C.glutamicum SYPA5-5高产L-精氨酸培养基为基础进行重组菌产酶优化。最优转化条件为：L-谷氨酰胺：乙胺为1：3，酶量为0.06 U/mL。采用底物流加策略高产L-茶氨酸，40 mL的转化体系包含：终浓度为0.9 U/mL的GGT，pH 10，37℃，从0 h开始每隔2 h补加20 mmol/L的L-谷氨酰胺，60 mmol/L的乙胺。[结果]C.glutamicumSYPA5-5/pXMJ19-ggt发酵上清液中GGT酶活达到（4.69±0.34）U/mL，C.glutamicum SYPA5-5/pXMJ19-Δsp ggt只检测到胞内酶活（0.99±0.17）U/mL，说明利用B.subtilis来源的信号肽可以实现GGT在C.glutamicum体系中分泌表达。最适产酶培养基条件为：葡萄糖浓度为10%；IPTG最适添加时间为0 h。批次流加在12 h时达到最大茶氨酸产量104.36 mmol/L，转化率为86.9%。[讨论]本文首次实现B.subtilis来源的γ-谷氨酰转肽酶基因（ggt）在C.glutamicum SYPA5-5中分泌表达，通过分批流加底物获得目前报道的利用重组C.glutamicum合成L-茶氨酸的最高产量。

Abstract:

[Objective] To construct a Corynebacterium glutamicum strain system for L-theanine production by the secretion expression of γ-glutamyltranspeptidase. [Methods] 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. [Results] 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%. [Conclusion] 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.