双拷贝tylD、tylF和tylJ弗氏链霉菌工程菌株构建及其发酵性能研究

以弗氏链霉菌工业生产菌株基因组DNA为模板,扩增泰乐星生物合成及组分转化关键基因tylD、tylF和tylJ,构建双拷贝tylD、tylF、tylJ弗氏链霉菌工程菌株。通过摇瓶发酵验证了工程菌株泰乐菌素发酵效价,并对筛选的一株工程菌利用荧光定量PCR检测其tylD、tylF和tylJ基因表达情况。研究结果表明,双拷贝tylD、tylF、tylJ弗氏链霉菌工程菌株泰乐菌素摇瓶发酵效价较出发菌株最高提高了28.1%,该菌株tylD、tylF、tylJ表达水平高于出发菌株,并且在进入稳定期后期表达量达到最大值。构建双拷贝tylD、tylF、tylJ弗氏链霉菌工程菌株可解除泰乐菌素合成限速环节,大幅度的提高泰乐菌素产量。

Construction of a double-copy tylD,tylF and tylJ Streptomyces fradie strain and evaluation of its fermentation performance

Objective The key genes of tylosin biosynthesis of tylD, tylF and tylJ were amplified with genomic DNA of Streptomyces fradiae industrial strain as PCR template, and double-copy tylD, tylF and tylJ Streptomyces fradie strain was constructed. The tylosin potency of engineered strains was verified by the shake flask fermentation. The expression of tylD, tylF, and tylJ of screened strains were detected by the way of real-time fluorescence quantitative PCR. The results showed that the highest increase of flask fermentation potency of engineering stains reached to 28.1% compared with that of the initial strain. Expression of tylD, tylF and tylJ genes in the engineering strain were higher than those of the initial strain and reached to the maximum in stationary phase. By constructing a double-copying tylD, tylF, and tylJ Streptomyces faecalis strains, the speed-limiting in the synthesis of tylosin can be removed, which could greatly improve the production of