小白链霉菌ε-聚赖氨酸降解酶生理功能分析

Physiological function analysis of ε-poly-L-lysine-degrading enzymes in Streptomyces albulus

摘要:

[目的] 研究小白链霉菌（Streptomyces albulus）中ε-聚赖氨酸降解酶（Pld）的分布特征和生理功能。[方法] 利用生物信息学手段对已报道的ε-聚赖氨酸（ε-PL）产生菌的Pld进行挖掘和分析，再通过遗传学方法对小白链霉菌M-Z18基因组中存在的两种pld进行敲除、回补和过表达，最后研究重组菌降解ε-PL能力、最小ε-PL抑制浓度（MIC）及其合成ε-PL情况。[结果] PldⅠ和PldⅡ广泛且同时分布于小白链霉菌中，蛋白序列高度保守；PldⅠ、PldⅡ在小白链霉菌M-Z18中均能行使降解ε-PL的功能，但PldⅡ降解活性占主导地位且PldⅠ和PldⅡ对降解ε-PL具有协同作用；pldⅠ、pldⅡ过表达重组菌对ε-PL的MIC值显著提高，其中双过表达pldⅠ和pldⅡ菌株对ε-PL的MIC值是出发菌株的2.19倍。构建的pld重组菌与出发菌株相比，在考察pH值范围内（pH 3.0-5.5）的ε-PL产量未表现出显著差异。[结论] 小白链霉菌中广泛分布PldⅠ和PldⅡ且序列高度保守，主要生理功能是保护小白链霉菌在中性环境中免受自身产物ε-PL的抑制。

Abstract:

[Objective] The distribution and physiological function of ε-poly-L-lysine-degrading enzyme (Pld) in Streptomyces albulus were investigated in this study. [Methods] The sequence of Pld in the reported ε-poly-L-lysine (ε-PL) producing strains were mined and analyzed from their genome by bioinformatics methods, and then two types of Plds in S. albulus M-Z18 genome were knocked out, complemented and overexpressed by genetic methods, those recombinant strains were used to study the degradation of ε-PL, the minimum inhibitory concentration (MIC) of ε-PL and their ε-PL productions. [Results] pldⅠ and pldⅡ are widely distributed in S. albulus, and their protein sequences are highly conserved. The results showed that PldⅠ and PldⅡ in the S. albulus M-Z18 all could degrade ε-PL. However, the degradation activity of PldⅡ was dominant, while PldⅠ and PldⅡ had a synergistic effect on the degradation of ε-PL. The MIC values of recombinant strains with pldⅠ or/and pldⅡ overexpression toward ε-PL were significantly increased, especially the MIC value of pldⅠ and pldⅡ co-overexpressed strain was 2.19 folds higher than that of the original strain. Surprisingly, these recombinant strains showed no significant difference in the production of ε-PL at pH 3.0-5.5 compared with the original strain. [Conclusion] PldⅠ and PldⅡ are highly conserved in S. albulus, which physiological functions are protect S. albulus from the inhibition of its metabolite ε-PL by degradation at the neutral pH.