Functional Study Of Genes Involved In Lomofungin Biosynthesis In Streptomyces Lomondensis S015

Streptomyces lomondensis S015,a strain isolated from the rhizosphere soil in the suburb of Shanghai,China,could biosynthesize an olive-yellow phenazine antibiotic lomofungin.Previous studies have identified the lomofungin biosynthesis gene cluster lphzGFEDCB(NCBI : KP721214.1)and a flavin-dependent monooxygenase gene lomoB that is responsible for C-7 hydroxylation of lomofungin in S.lomondensis S015.The lomoB gene deletion mutant lost the ability to biosynthesis lomofungin and only produced another phenazine compound 1-carbomethoxy-6-formy-4,9-dihydroxy-phenazine(CMFDP).To further clarify the biosynthesis pathway of lomofungin,this study focuses on the function of an oxidoreductase gene lomoF,an acyl-coA dehydrogenase gene lomoA and a methyltransferase gene lomoC on lomofungin biosynthesis in S.lomondensis S015.Gene lomoF was inactivated by in-frame partial deletion and the fermentation metabolites from the mutant strain were analyzed by HPLC compared with that from the wild-type strain S015.The production of lomofungin in the gene deletion mutant strain decreased to about 60% of that in the wild-type strain S015.Meanwhile,an unknown compound A was also detected in the mutant strain.Further investigation of the structure of compound A was performed by LC-MS.The molecular mass of compound A was detected to be 298.25 Da,in accordance with CMFDP isolated form gene lomoB deletion mutant.Further HPLC-MS-MS analysis showed that the fragments from compound A and compound CMFDP were exactly the same,which suggested that compound A is CMFDP.The lomoF complementary mutant restored lomofungin production level without other phenazine derivatives,while lomoF overexpression mutant produced more lomofungin,appropriately 1.5 times compared with that from S015 wild-type.This result indicated that LomoF could promote the hydroxylation on the C-7 position of lomofungin.Inactivation of gene lomoA and lomoC by in-frame partial deletion in S015 resulted in the gene deletion mutant strains.HPLC analysis showed that these two mutant strains were not able to generate lomofungin,and no other phenazine derivatives were detected either.The lomoA and lomoC complementary mutants restored the ability to produce lomofungin.These results confirmed that lomoA and lomoC play important roles in lomofungin biosynthesis,and their detailed functions remain to be explored in future studies.This study focused on the influence of gene lomoF,lomoA and lomoC on lomofungin biosynthesis.LomoF was indicated to promote the hydroxylation on the C-7 position and LomoA and LomoC play important roles in the lomofungin biosynthesis pathway.The results of this study lay a theoretical foundation for further investigation of lomofungin biosynthesis in Streptomyces.