天蓝色链霉菌孢子色素whiE在大肠杆菌中的异源生物合成

Heterologous biosynthesis of spore pigment whiE from Streptomyces coelicolor in Escherichia coli

摘要:

[目的] 在大肠杆菌中完整重构孢子色素whiE的生物合成途径，分离纯化表达体系中合成的新化合物，并解析whiE的生物合成途径。[方法] 构建whiE-ORFII、whiE-ORFVII和whiE-ORFI的单基因重组质粒，SDS-PAGE检测蛋白表达情况；借助Xba I与Spe I互为同尾酶的特性，实现多基因组合串联；构建好的重组质粒再导入大肠杆菌菌株BAP1中进行异源表达，并用高效液相色谱（HPLC）检测发酵产物；依次使用正相硅胶柱和反向半制备柱分离发酵产物，四级杆飞行时间质谱仪（Q-TOF MS）鉴定发酵产物分子量。[结果] whiE-ORFII、whiE-ORFVII和whiE-ORFI均获得可溶性表达；这3个基因单个串联到菌株BTw95中均未检测到新的产物生成；而whiE-ORFII和whiE-ORFVII、whiE-ORFI和whiE-ORFVII双基因组合以及三基因组合串联到BTw95中可检测得到两种化合物ZYC-1和ZYC-2。在负离子模式下进行Q-TOF MS检测，ZYC-1的[M-H]^-为419.0748，推测分子式为C₂₃H₁₆O₈；ZYC-2的[M-H]^-为465.0743，推测分子式为C₂₄H₁₈O₁₀。[结论] 本研究推进了孢子色素whiE生物合成途径在大肠杆菌中的异源重构，分离鉴定了2个十二酮II型聚酮化合物，并推测了孢子色素whiE的生物合成途径。

Abstract:

[Objective] Completely reconstruction of the biosynthetic pathway of whiE in Escherichia coli, isolation and purification of new compounds synthesized in the recombinant strain for elucidation of the biosynthetic pathway of whiE.[Methods] Construction of single-gene recombinant plasmids of whiE-ORFII, whiE-ORFVII, whiE-ORFI, and SDS-PAGE analysis of the fusion protein expression; isoschizomers Xba I/ Spe Ⅰ were adopted to assemble multiple genes in a single plasmid construct. The recombinant plasmid was then introduced into Escherichia coli BAP1 for heterologous expression, and the fermentation products was detected by high performance liquid chromatography (HPLC); the normal phase silica gel column and the reverse semi-preparative column were used sequentially to separate the fermentation products, the quadrupole high resolution mass spectrometry (Q-TOF MS) was used to identify the molecular weight of the fermentation products.[Results] whiE-ORFII, whiE-ORFVII and whiE-ORFI were solubly expressed; no new product was detected when these three genes were added to the strain BTw95 individually; two compounds ZYC-1 and ZYC-2 can be detected in the strains coupled with whiE-ORFII & whiE-ORFVII, whiE-ORFI & whiE-ORFVII as well as all the three genes. Q-TOF MS analysis in negative ion mode showed that the[M-H]^- of ZYC-1 is 419.0748 with a proposed molecular formula, C₂₃H₁₆O₈ and[M-H]^- of ZYC-2 is 465.0743 with a proposed molecular formula, C₂₄H₁₈O₁₀.[Conclusion]] This study achieved the heterologous reconstruction of the spore pigment whiE biosynthetic pathway in E. coli. Two dodecaketide type II polyketides were identified and isolated. The biosynthetic pathway of the spore pigment whiE was also proposed.