糖多孢红霉菌SACE_Lrp调控分支氨基酸氨基转移酶IlvE的分子机制研究

Lrp家族是一类广泛存在于原核生物中的转录调控因子，能够参与调控细胞的多个生理过程。分支氨基酸是聚酮类抗生素生物合成前体的重要来源，对抗生素的合成产量至关重要。申请者前期的研究已经证实，糖多孢红霉菌中Lrp家族调控因子SACE_Lrp(SACE_5388)的缺失能够显著影响细胞内分支氨基酸的代谢转化。进一步分析发现SACE_Lrp间接调控分支氨基酸氨基转移酶ilvE基因的转录，但是具体的调控机制并不清楚。本项目拟通过转录组分析联合蛋白保守结合位点的全基因组预测，利用qRT-PCR、EMSA、体内GFP报告系统、ITC和基因敲除等技术，建立SACE_Lrp与其他调控因子之间的级联调控关系，最终解析SACE_Lrp对分支氨基酸氨基转移酶IlvE的分子调控机制。本项目为深入认识糖多孢红霉菌中红霉素生物合成调控网络及分子机制，及通过基因工程手段改造分支氨基酸代谢过程提高抗生素产量奠定基础。

Leucine-responsive regulatory proteins (Lrps) are a group of transcriptional regulators that regulate diverse cellular processes in bacteria and archaea. Branched chain amino acid (BCAA) degradation is a major source of the precursor for polyketide biosynthesis, and is crucial for the production of antibiotics. In a recent report, we have confirmed that the deletion of the SACE_Lrp (SACE_5388, encoding leucine-responsive regulatory protein) significantly increased the catabolism of intracellular BCAAs in Saccharopolyspora erythraea. We found that SACE_Lrp indirectly regulated the transcription of ilvE gene encoding BCAA aminotransferase, but the corresponding molecular mechanism was not clear. In this proposal, based on inactivation of SACE_Lrp, we will search for the downstream regulatory genes of SACE_Lrp by transcriptomic analysis and qRT-PCR, and further for its direct target genes by EMSA (electrophoretic mobility shift assay) and in vivo green fluorescent protein (GFP) report system, constructing the cascade regulatory relationship between SACE_Lrp and other regulatory proteins. Our aim is to illustrate the molecular regulatory mechanism of SACE_Lrp regulating the BCAA aminotransferase IlvE in S. erythraea. This project would be available for the understanding the regulatory network of erythromycin biosynthesis and subsequent enhancement of erythromycin production with genetic engineering of BCAA degradation pathway in S. erythraea.