染色体DNA琼脂糖包埋法辅助的ExoCET技术克隆放线菌天然产物生物合成基因簇

Agarose-embedded chromosomal DNA combines with ExoCET technology to capture the biosynthetic gene clusters of natural products in Actinobacteria

摘要:

【目的】本研究旨在通过将琼脂糖包埋染色体DNA的方法与ExoCET重组技术相结合，建立放线菌天然产物生物合成基因簇的捕获方法。然后将克隆基因簇导入通用底盘宿主中，实现目标生物合成基因簇的异源表达。【方法】首先，利用低熔点琼脂糖包埋技术制备菌株的染色体基因组总DNA，再用限制性内切酶消化含有染色体DNA的琼脂块，获得线性化的DNA样品；然后利用ExoCET重组技术，以p15A线性载体片段将目标基因簇线性片段进行捕获；再通过PCR-targeting的方法向目标质粒中引入所需的接合转移DNA元件。接着，将改造质粒通过接合转移导入到Streptomyces coelicolor M1252宿主中，获得不同的重组菌株。最后，对不同的菌株进行发酵并提取化合物，最后进行活性检测以及质谱检测。【结果】通过该方法，从菌株S.lincolnensisNRR2936中成功获得了林可霉素生物合成基因簇(lmb-BGC)，从菌株Nonomuraea nitratireducens WYY166^T中克隆得到了2个核糖体肽类化合物的生物合成基因簇(nioblantin，niob-BGC和nitblantin，nitb-BGC)，并实现了lmb-BGC在天蓝色链霉菌M1252中的成功表达。【结论】本研究通过将低熔点琼脂糖包埋技术与ExoCET重组技术进行合理整合，定向克隆得到了林可霉素以及2个新颖的羊毛硫肽类化合物的生物合成基因簇。然后，分别对重组质粒改造后，在天蓝色链霉菌M1252宿主中进行表达，分别获得重组菌株MJX01、MJX02和MJX04。最后，利用质谱以及活性测试的手段对发酵提取物进行了检测，确定了林可霉素生物合成基因簇在天蓝色链霉菌M1252中成功表达。本研究为通过基因簇克隆和异源表达发掘新化合物奠定了基础。

Abstract:

[Objective] To develop a method of cloning biosynthetic gene clusters of natural products (NP-BGCs) by combining agarose-embedded chromosomal DNA strategy with exonuclease combined with RecET recombination (ExoCET) technology and transform the cloned gene clusters into chassis cells for the expression of target NP-BGCs in heterologous hosts.[Methods] Firstly, the chromosomal DNA of the targeted strain was prepared by agarose-embedded plugs with the low-melting-temperature agarose and digested with the restriction enzymes to yield the linear DNA sample. Then, the linear target BGC was captured by the linear vector of p15A through the ExoCET technology. The desired integrative and conjugative elements were introduced into the BGC-containing plasmid through PCR-targeting approach. Subsequently, the final modified plasmid was introduced into Streptomyces coelicolor M1252 by intergeneric conjugation to yield the desired recombinant strains. Finally, the recombinant strains were fermented and analyzed for target compound production by UPLC-ESI-MS and the inhibitory activity against different indicator strains was detected. [Results] With this method, the BGC of lincomycin (lmb-BGC) and the BGCs of two ribosomal peptides (nioblantin, niob-BGC and nitblantin, nitb-BGC) were obtained from S.lincolnensis NRR2936 and Nonomuraea nitratireducens WYY166^T, respectively. Finally, the lmb-BGC was expressed in M1252 for production of lincomycin. [Conclusion] In this study, the lmb-BGC and two novel lanthipeptide BGCs were cloned by the agarose-embedded chromosomal DNA in combination with ExoCET technology. Then, the BGC-containing plasmids were modified for conjugations. The recombinant strains MJX01, MJX02, and MJX03 were obtained by conjugation with the strain M1252 host. The fermentation broth was extracted and analyzed by UPLC-ESI-MS and the anti-bacterial activity was detected. Finally, our results revealed that the lincomycin was successfully produced in the strain M1252 containing the lmb-BGC. This study lays the foundation for the discovery of new compounds through gene cluster cloning and heterologous expression in the chassis strain.