红色糖多孢菌A226-ΔeryCⅢ突变体的构建及产物分析

Construction of Saccharopolyspora erythraea A226-ΔeryCⅢ mutant and identification of its product

目的构建红色糖多孢菌糖基转移酶EryCⅢ失活突变体,为探索红霉素糖基结构修饰和合成新型红霉素衍生物打下基础。方法通过PCR扩增方法将eryCⅢ基因缺失130bp后,克隆至同源重组型质粒pWHM3上,构建pWHM3-ΔeryCⅢ,将其导入红色糖多孢菌A226中。经过两次同源重组,筛选出1株缺失EryCⅢ酶活性的红色糖多孢菌A226-ΔeryCⅢ突变体。使用抑菌实验、薄层层析和质谱对其发酵产物进行分析,并通过回补实验进一步验证。结果与结论对红色糖多孢菌A226-ΔeryCⅢ染色体序列测定表明,eryCⅢ基因相应位点缺失130bp,突变菌株发酵产物对枯草芽孢杆菌无抑制作用,薄层和质谱结果表明突变菌株积累3-L-mycarose红霉内酯B(MEB),而并没有发现红霉素产物。回补验证发现eryCⅢ基因导入A226-ΔeryCⅢ使得其恢复了红霉素合成能力。所构建的红色糖多孢菌EryCⅢ失活突变体A226-ΔeryCⅢ,为探讨组合合成红霉素衍生物奠定基础。

Objective To construct a glycosyltransferase EryCⅢ inactivated mutant of Saccharopolyspora erythraea in order to study how to modify sugar structures and synthesize novel derivatives of erythromycin.Methods The mutant DNA fragment of eryCⅢ gene after deletion of a 130 bp in key site was amplified by PCR and cloned into the homologous recombinant vector pWHM3 to form pWHM3-ΔeryCⅢ,and then pWHM3-ΔeryCⅢ was transferred into Sac.erythraea A226.After two rounds of homologous recombination,one mutant lacking EryCⅢ activity named A226-ΔeryCⅢ was screened out.Bioassay test,TLC and MS were used to analyze the fermentation products of A226-ΔeryCⅢ,and complementation test was used for verification.Results Sequence analysis of Sac.erythraea mutant A226-ΔeryCⅢ showed that the corresponding 130 bp length fragment in eryCⅢ gene was deleted,and bioassay test indicated that the fermentation product of A226-ΔeryCⅢ did not have the ability to inhibit Bacillus subtilis.The results of TLC and MS showed that there was 3-L-mycarose erythronolide B（MEB）accumulated in the mutant,but no erythromycins.The complementation test suggested that introduction of eryCⅢ gene into A226-ΔeryCⅢ restored the ability to produce erythromycins.Conclusion The Sac.erythraea mutant A226-ΔeryCⅢ with glycosyltransferase EryCⅢ inactivated is successfully constructed,and it sheds light on synthesis of erythromycin derivatives through combinatorial biosynthesis.