Halomonas socia NY-011^T中四氢嘧啶合成基因簇ectABC和ectD的克隆及其功能鉴定

Cloning and characterization of ectABC and ectD from Halomonas socia NY-011~T

由ectA、ectB、ectC和ectD编码的酶可以催化对细胞起保护作用的四氢嘧啶类物质的生物合成,嗜盐菌新种Halomonas socia NY-011~T ectABC和ectD为四氢嘧啶和羟基四氢嘧啶的生产提供新的基因来源.通过SEFA-PCR步移方法从Halomonas socia NY-011~T中克隆ectA、ectB、ectC和ectD基因,并分别构建ectABC和ectD的原核表达质粒,在E.coli BL21(DE3)中异源表达,利用SDS-PAGE对重组蛋白进行鉴定,通过ACQUITY UPLC MS/MS检测四氢嘧啶和羟基四氢嘧啶的生成.结果显示:H.socia NY-011~T的四氢嘧啶合成基因ectA、ectB、ectC形成基因簇ectABC(KP717055-57),大小为2 506 bp,四氢嘧啶羟化酶基因ectD大小为942 bp(KP717058),与近源种H.elongata DSM258同源性分别为81%和78%;SDS-PAGE检测到4种融合蛋白,大小(M_r)分别为21.6×10~3、46.4×10~3、14.4×10~3、55.3×10~3,与预期相符;利用ACQUITY UPLC MS/MS发现仅表达ectABC的重组菌株[E.coli BL21(DE3)/pltac-ABC]中只存在四氢嘧啶,而在表达ectABC和ectD的重组菌株[E.coli BL21(DE3)/pltac-ABC/pETect D]中检测到四氢嘧啶和羟基四氢嘧啶.本研究从H.socia NY-011~T中分离得到的四氢嘧啶类物质的合成基因,能够在E.coli BL21(DE3)中异源表达并行使功能,为四氢嘧啶类物质异源生产提供了新的基因来源.

Ectoines can protect cells from extreme environment through stabilizing protein and nucleic acid. The process ectoine and hydroxyectoine synthesized is catalyzed by four enzymes encoded by ectA, ectB, ectC and ectD. Halomonas socia NY-011^T provides new gene for ectoine and hydroxyectoine synthesis. This study aimed to clone and characterize ectA, ectB, ectC and ectD of Halomonas socia NY-011^T. SEFA-PCR（self-formed adaptor PCR） was firstly used for cloning ectA, ectB, ectC and ectD from Halomonas socia NY-011^T, then ectABC and ectD were introduced into prokaryotic expression vector; after heterologous expression in E. coli BL21（DE3）, recombinant proteins were detected by SDS-PAGE. Ectoine and hydroxyectoine were authenticated by ACQUITY UPLC MS/MS finally. Ectoine biosynthetic enzymes gene cluster ectABC was 2506 bp（KP717055-57） in length and ectoine hydroxylase gene ectD was 942 bp（KP717058）, both conserved to their allied species（81% and 78%）. The four fusion proteins were detected by SDS-PAGE, the sizes of which were in accordance with expectation（M_r 21.6 × 10^3, 46.4 × 10^3, 14.4 × 10^3, 55.3 × 10^3, respectively）. Using ACQUITY UPLC MS/MS, both ectoine and hydroxyectoine were detected in E. coli BL21（DE3）/pltac-ABC/pETect D, and only ectoine was found in E. coli BL21（DE3）/pltac-ABC. This study cloned ectABC genes and ectD from Halomonas socia NY-011^T and heterologously expressed in E. coli BL21（DE3）. Ectoine and hydroxyectoine were detected by UPLC in E. coli BL21（DE3）/pltac-ABC/pETect D, providing new gene for ectoines synthesis.