The Study Of The Pathway-specific Regulatory Mechanism In The Tacrolimus Biosynthetic Pathway

Tacrolimus(FK506) is a 23-membered polyketide macrolide with immuno-suppressant activity that was produced by Streptomyces, and was used clinically for treating the rejection after organ transplantation of liver, kidney, pancreas, and heart. We found two transcriptional regulators fkbN and tcs7 in the tacrolimus biosynthetic gene cluster of Streptomyces tsukubaensis L19, an industrial strain producing tacrolimus.Firstly, we analyzed the 8 transcription units of tacrolimus cluster through reverse transcription-PCR. The eights transcription units were tcsA-tcsB-tcsC-tcsD; tcs1; tcs2-tcs3-tcs4-tcs5; fkbG;fkbB-fkbC-fkbL-fkbL-fkbJ-fkbJ-fkbI; fkbO-fkbP-fkbA-fkbD-fkbM; tcs6-fkbQ-fkbN, tcs7. Overexpression of fkbN and tcs7 improved the production of tacrolimus about 76% and 40%, respectively. It suggested that FkbN and Tcs7 were both pathway-specific positive regulators.Then, Comparison of the transcription level of wildtype L19 and overexpression of fkbN strain L21 revealed that the expression level of all the target genes was greatly up-regulated. FkbN belongs to LAL family transcriptional regulators. Alignment of different LAL proteins revealed FkbN may possess ATPase activity and be affected by ATP. Analysis of binding targets by EMSA using the purified FkbN-DBD proteins revealed that FkbN-DBD specifically bound to four regions to activate the biosythesis of tacrolimus:tcsAp, fkbGp, the intergenic region of fkbB-fkbO and the intergenic region of tcs6-tcs7. These regions were responsible for the transcription of tcs7, important PKS/NRPS genes, and genes involved in precursor biosynthesis. Also, results of DNase I footprinting revealed that all the binding sequences covered a pair of reverse complements about 10bp.The other regulator Tcs7 belongs to LysR family transcriptional regulators. Alignment analysis of Tcs7 showed a 35.3% sequence identity with nine additional LysR proteins. According to the connection structure and function, we speculated that Tcs7 may have the function of autoregulation. Comparison of the transcription level of wildtype L19 and overexpression of tcs7 strain L22 revealed that the expression level of most of the target genes was up-regulated, including the transcription level of fkbN. Analysis of the binding targets of Tcs7 by EMSA revealed Tcs7-DBD didn’t bind to the biosynthetic genes, but the intergenic region of tcs6-tcs7. This region contains two promoters, responsible for the transcription of tcs7 and tcs6-fkbQ-fkbN, suggested Tcs7 may affect the biosynthesis of tacrolimus by regulating the transcription of fkbN directly.On the basis of the above results, we recombined the pathway-specific pathway through combinative overexpression of tcs 7 and fkbN, the production of the resulting strain L23 increased by 89.3% in comparison with wild type L19.In conclusion, we proposed the pathway-specific regulatory model of the biosythesis of tacrolimus. FkbN positively regulated the transcription of tcs7 and main biosythetic genes, which may be enhanced by ATP. However, Tcs7 also could positively regulate the transcription of fkbN, thus forming a self-amplifying positive regulating circuit. We speculated this circuit was limited because of the autoregulation of Tcs7 and the regulation of other transcriptional regulators to maintain the balance of the biosythesis of tacrolimus.