林可链霉菌NRRL 2936中帕马霉素生物合成基因簇的异源表达及调控基因功能研究

Heterologous expression of the biosynthetic gene cluster of pamamycin and investigation on the functions of regulatory genes from Streptomyces lincolnensis NRRL 2936

【背景】帕马霉素属于大环内酯类抗生素,具有较好的抗感染活性。该类化合物独特的化学结构和显著的生理活性受到了许多研究者的关注。同时,本实验室在林可链霉菌NRRL2936的全基因组序列中发现了帕马霉素的生物合成基因簇。【目的】尽管其生物合成途径已经得到了解析,但其生物合成基因簇中的2个调控基因功能尚不清楚。本研究从林可链霉菌NRRL2936的基因组文库中克隆了含有帕马霉素生物合成完整基因簇的质粒pJQK450,开展了质粒pJQK450在链霉菌中的异源表达,实现了帕马霉素的异源合成,并初步确定该生物合成基因簇中两个调控基因的功能。【方法】利用聚合酶链式反应递缩基因组文库筛选的方法,从林可链霉菌(Streptomyces lincolnensis)NRRL 2936基因组文库中筛选到了含有帕马霉素生物合成完整基因簇的Fosmid质粒pJQK450。然后,将该质粒转化到E.coli ET12567/pUZ8002中,利用大肠杆菌-链霉菌双亲接合转移的方法将pJQK450转入异源宿主中。对获得的异源表达菌株进行发酵产物的制备,采用耻垢分枝杆菌mc2155作为指示菌株进行帕马霉素生物活性测试,并结合LC-MS的分析确定帕马霉素的产生情况。最后,通过基因失活与回补的方法,考察帕马霉素生物合成基因簇中调控蛋白PamR1和PamR2对帕马霉素生物合成的影响。【结果】帕马霉素生物合成基因簇在天蓝色链霉菌M1154中实现了表达,证明PamR1和PamR2负调控了帕马霉素生物合成的过程。【结论】帕马霉素完整基因簇的成功异源表达,一方面便于其生物合成途径的遗传改造,为帕马霉素的生物合成及优产改造研究奠定了基础;另外,调控基因功能的研究为帕马霉素的产量优化提供了改造的目标。

[Background] Pamamycins are macrolid compounds with anti-infectin bioactivities. Their unique structural features and promising biological activity stimulated extensive efforts towards their various investigations. Furtehermore, we discovered that the biosynthetic gene cluster of pamamycins was located in the chromosome of Streptomyces lincolnensis NRRL 2936. [Objective] The biosynthetic pathway had been investigated, and the function of two regulatory genes in the gene cluster was elusive. In this research, the plasmid pJQK450 containing a full length of pamamycin biosynthetic gene cluster was cloned from the genomic library of S. lincolnensis NRRL 2936 and introduced into heterologous hosts by intergeneric conjugation. Subsequently, the function of two regulatory genes was elucidated by targeted gene disruption and complementaion. [Methods] The Fosmid, named pJQK450, containing of the completed pamamycin gene cluster was captured by narrow-down polymerase chain reaction strategy from Streptomyces lincolnensis NRRL 2936 Fosmid genome library. Plasmid pJQK450 was transferred into the E. coli ET12567/pUZ8002 and then introduced into the heterologous hosts by intergeneric conjugation. Then, the validated-exconjugants were fermented, and the biosynthetic capacity of pamamycin of the recombinant strains was evaluated by bioactivity analysis against Mycobacteriun smegmatis mc2155 and LC-MS detection. Furthermore, the regulation roles of PamR1 and PamR2 were determined by targeted gene inactivation and complementation experiments. [Results] The entire gene cluster of pamamycin was successful expressed in S. coelicolor Ml154. Through genetic validation, both PamR1 and PamR2 were found to be negative regulators in pamamycin biosynthesis. [Conclusion] Heterologous expression of pamamycin facilitates the biosynthesis engineering and targeted accumulating pamamycin component. Additionally, the determination of the two negative regulatory genes paved way for the yield improvement of pamamycin.