糖多孢红霉菌中与红霉素合成相关调控因子间的干扰机制研究

Saccharopolyspora erythraea, which could biosynthesis of erythromycin, was important for pharmaceutical industry and synthetic biology. Combinational deletion or overexpression multiple regulatory genes was widely used for improvement production of antibiotics. However, effects raised by combination of regulatory factors were very complicated. In our previous study, we have reported that, deletion of negative regulatory genes SACE_3446 or SACE_3986 could increase erythromycin production in S. erythraea, and double deletion of them could further enhance erythromycin production. Additionally, overexpression of positive regulatory gene SACE_7301 could also increase erythromycin production in S. erythraea. Unexpectedly, when we overexpressed SACE_7301 in S. erythraea with both deletion of SACE_3446 and SACE_3986, erythromycin production was obviously decreased. These results indicated that, interference was raised by combination of regulatory factors SACE_3446, SACE_3986 and SACE_7301. However, the interference mechanism remains largely unknown. In this project, first, we would evaluate the function of SACE_3446, SACE_3986 and SACE_7301 in the interference with various combination of these regulatory factors. Then, candidate genes, located downstream of the three regulatory factors and involved in the interference mechanism, would be identified with transcriptomic analysis, gSELEX (genomic systematic evolution of ligands by exponential enrichment), gene deletion, EMSA (Electrophoretic Mobility Shift Assay) and ITC (Isothermal Titration Calorimetry). Based on these research, we would dissect the interference mechanism. This project would be available for the research on effects raised by combination of regulatory factors and subsequent enhancement of erythromycin production with genetic improvement of S. erythraea.

糖多孢红霉菌是红霉素产生菌，在医药与合成生物学领域具有重要价值。选择多个调控因子共敲除或过表达是提高抗生素产量的一种常用方法，然而，调控因子之间的组合效应很复杂。本课题组已报道，敲除糖多孢红霉菌负调控基因SACE_3446或SACE_3986，可提高红霉素产量，双敲可使产量进一步提高，单独过表达正调控基因SACE_7301也可提高红霉素产量。然而在双敲SACE_3446和SACE_3986突变株中过表达SACE_7301，红霉素产量却反而显著下降，表明SACE_3446、SACE_3986与SACE_7301之间存在干扰效应，但其干扰机制还不清晰。本项目首先对上述调控因子设计不同组合以鉴定它们在干扰效应中的作用；再利用转录组分析、gSELEX、基因敲除、EMSA、ITC等技术研究下游基因在干扰效应中的功能，最终解析干扰机制。本项目有助于调控因子的组合效应研究及后续遗传改良提高红霉素产量。

调控因子组合改造是获得高产菌株的一个重要策略，然而调控因子之间组合效应十分复杂。本课题组前期报道，敲除糖多孢红霉菌负调控基因SACE_3446或SACE_3986，可提高红霉素产量，双敲可使产量进一步提高，单独过表达正调控基因SACE_7301也可提高红霉素产量。然而在双敲SACE_3446和SACE_3986突变株中过表达SACE_7301，红霉素产量却反而显著下降，表明SACE_3446、SACE_3986与SACE_7301之间存在干扰效应，但其干扰机制还不清晰。本课题在单敲除SACE_3446、SACE_3986菌株中分别过表达SACE_7301，前者红霉素产量无明显变化，而后者产量下降约26%。对ΔSACE_3986/A226、ΔSACE_7301/A226进行转录组测序，寻找下游转录变化方向一致的基因作为候选干扰因子。结果显示，共有104个候选基因，其中转录调控因子13个。qRT-PCR及EMSA结果表明，SACE_7301直接抑制SACE_1409和SACE_6211的转录，而SACE_3986对它们行使间接抑制调控作用。过表达SACE_1409导致红霉素产量相比A226下降36%，过表达SACE_6211则下降58%。EMSA结果显示，SACE_1409结合红霉素合成基因簇中eryAI和ermE启动子区，而SACE_6211不结合，表明SACE_1409可直接抑制红霉素的生物合成，SACE_6211起间接抑制作用。该项研究表明，SACE_1409、SACE_6211是SACE_7301和SACE_3986之间干扰效应的干扰因子。本项研究有利于后续调控网络的优化和遗传改良提高红霉素产量，对其他抗生素菌株的遗传改良也具有一定借鉴意义。