东方拟无枝酸菌中基因AORI_2943的敲除对ECO-0501合成的影响

摘要

目的 对位于ECO-0501生物合成基因簇内AORI_2943基因进行功能鉴定,进一步解析东方拟无枝酸菌中ECO-0501的生物合成途径.方法 通过同源重组和定点整合的方式构建AORI_2943缺失、回补及过表达突变株.高效液相及质谱分析突变株发酵产物的变化,分析AORI_2943在 ECO-0501合成中的作用.结果 AORI_2943缺失突变株不产ECO-0501和去甲基ECO-0501,只产生结构上缺失了C5N单位(2-氨基-3-羟基环戊-2-烯酮)的前体物质a和b,发酵液中未检测出C5N单位.对缺失突变株回补后,有少量ECO-0501及去甲基ECO-0501产生,同时仍然有大量前体化合物a和b的积累.在野生菌的基础上过表达AORI_2943,并未明显提高ECO-0501的产量.结论 AORI_2943是ECO-0501合成的关键基因,主要参与了ECO-0501前体化合物 C5N单位的合成.%Objective To determine the function of theAORI_2943gene located in the biosynthesis gene cluster of ECO-0501 from Amycolatopsis orientalis. Methods The disruption, complementation and overexpression ofAORI_2943 were conducted by homologous recombination and site-specified integration. The metabolites in the fermentation broth of the resulted mutants were analyzed by high performance liquid chromatography and mass spectrometry. Results Disruption ofAORI_2943 inAmycolatopsis orientalis resulted in the disappearance of ECO-0501 and demethyl-ECO-0501 and the appearance of two new compounds a and b in the fermentation broth of the mutant. The two new compounds a and b, lacking the 2-amino-3-hydroxy-cyclopent-2-enone moiety (C5N) in ECO-0501, were determined as the precursors of ECO-0501 and demethyl-ECO-0501, respectively. Complementation of the disrupted mutant with intactAORI_2943 restored the ECO-0501 production. However, overexpression ofAORI_2943 did not improve the production of ECO-0501. ConclusionAORI_2943 is crucial for ECO-0501, specifically the C5N unit biosynthesis. To improve the production of ECO-0501, overexpression ofAORI_2943 alone is not enough.