Assembly and heterologous expression of the pholipomycin biosynthetic gene cluster

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.