福林霉素生物合成基因簇的组装和异源表达

Assembly and heterologous expression of the pholipomycin biosynthetic gene cluster

摘要:

【目的】本研究旨在以来源于林可链霉菌NRRL2936中的nosokomycin B₂的生物合成基因簇（noso-BGC）为基础，组装获得完整的福林霉素生物合成基因簇（pho-BGC），再利用异源表达策略，激活pho-BGC的表达并通过底盘宿主的优选实现福林霉素发酵产量的提升。【方法】首先，在林可霉素基因簇缺失突变株JCK126基因组中，整合了福林霉素生物合成所缺失的moeA4、moeB4和moeC4 3个环合成基因（来源于加纳链霉菌ATCC 14672），通过对重组菌株LX19的发酵和提取物的检测确认了pho-BGC在林可链霉菌中的沉默表达。然后，利用基因组装技术将moeA4、moeB4和moeC4 3个环合成基因与noso-BGC进行连接，得到了含有完整pho-BGC的质粒pJQK572。接着，通过接合转移将质粒pJQK572分别导入Streptomyces coelicolor M1152、Streptomyces lividans SBT18、Streptomyces lividans LJ1018和Streptomyces coelicolor M1446宿主中获得不同的重组菌株LX20、LX21、LX22和LX23。最后，通过对不同重组菌株进行发酵并对提取物进行生物活性分析以及UPLC-TOF MS检测，确定福林霉素在不同异源宿主中的合成情况，并结合二级质谱分析（ESI-MS²）对其结构进行确定。【结果】pho-BGC在天蓝色链霉菌M1152中成功实现了异源表达，并在4拷贝天蓝色链霉菌M1446中发酵产量提高了约20%。【结论】本研究通过系统的发酵检测确定了福林霉素生物合成基因簇在林可链霉菌中是沉默的；然后，在nosokomycin B₂基因簇的基础上，构建了含有完整pho-BGC的质粒pJQK572；获得了pho-BGC在不同宿主中的异源表达产物后，利用多种液相-质谱联用技术对发酵提取物进行检测，确定了pho-BGC在天蓝色链霉菌M1152中成功表达，接着通过多拷贝整合技术在菌株LX23中将福林霉素的产量提高了约20%。完整pho-BGC的拼接和在菌株LX20 & LX23中的异源表达，为福林霉素生物合成机制的探索和高产优化奠定了基础。

Abstract:

[Objective] To assemble a complete pholipomycin biosynthetic gene cluster (pho-BGC) based on the nosokomycin B₂ biosynthetic gene cluster (noso-BGC) derived from Streptomyces lincolnensis NRRL2936, and activate pho-BGC through heterologous expression for production increase of pholipomycin using the screening of the chassis hosts.[Methods] Firstly, three genes moeA4, moeB4, and moeC4 were cloned from the genome of moenomycin producer Streptomyces ghanaensis ATCC 14672 and introduced into lincomycin gene cluster deletion strain JCK126, which yielded the recombinant strain LX19. The fermentation extract detection of LX19 showed that pho-BGC was silent in S. lincolnensis NRRL2936. Subsequently, the gene cassette carrying moeA4, moeB4, and moeC4 was inserted into noso-BGC by gene assembly, resulting in the plasmid pJQK572 containing the intact pho-BGC. Then, plasmid pJQK572 was introduced into Streptomyces coelicolor M1152, S. lividans SBT18, S. lividans LJ1018, and S. coelicolor M1446, which generated strains LX20, LX21, LX22, and LX23, respectively. Finally, the pholipomycin production ability of different recombinants was estimated by bioactivity analysis and UPLC-TOF MS of fermented extracts, and the chemical structure of pholipomycin was identified by ESI-MS².[Results] The complete pho-BGC achieved heterogeneous expression in S. coelicolor M1152. Moreover, the yield of pholipomycin was increased by 20% in S. coelicolor M1446 (carrying four copies of the ΦC31-attB sites).[Conclusion] This study determined that pho-BGC was silent in S. lincolnensis by systematic fermentation assay. Then, plasmid pJQK572 containing the complete pho-BGC was constructed based on noso-BGC. The liquid-mass spectrometry for the fermented extracts of different pho-BGC heterogeneous hosts determined that pholipomycin was successfully synthesized in S. coelicolor M1152. Moreover, the yield of pholipomycin in strain LX23 was increased by 20% through multi-copy integration of pho-BGC. The successful construction and heterologous expression of pho-BGC laid a foundation for revealing the biosynthesis mechanism and increasing the yield of pholipomycin.