The Physiology Function Of Lichenysin Produced By Bacillus Licheniformis And Construction Of High Yield Strain

Surfactin family synthesized by Bacillus is a kind of lipopeptide with strong surface activity,which has a great potential to apply for the fields of Microbial enhanced oil recovery,bioremediation,anti-viral medicine and so on.Due to its low-yield and high-costs of fermentation,application extension and research development of Lichenysin are limited.On the other hand,the role lipopeptides play in bacteria's lifecycle has not attracted sufficient attention.The main purpose of this study is to construct Lichenysin high-yield strains,and to study its regulating role in bacteria's growth.In this study,analysis of whole genome sequencing of Bacillus licheniformis WX-02 showed that this strain has complete gene elements for Lichenysin synthesis:a gene operon called lchA encoding synthase system and sfp encoding phosphopantetheinyl transferase(PPTase),PPTases can activate the synthase system.The broth of B.licheniformis WX-02 has oil spreading activity,so we extract and purify lipopeptide from the supernatant of fermentation broth.Extracts detected by HPLC-MS,revealing that B.licheniformis WX-02 mainly synthesize four Lichenysin G homologues,linked to different fatty acid chains with a length of C13,C14 and C15 respectively.The promoter of lchA(PlchA)is regulated by ComA?P,and its transcription initiation ability is so weak.In this study,using genetic engineering techniques,we replace PlchA by promoter P43.The construction strain was named WX-02 P43lchA.After cultivating those two strains in Cooper's minimal medium and detecting the relative content of Lichenysin by HPLC,we found the production of wild type WX-02 was 1249.6 ± 51.6 mAU×s,and the production of WX-02 P43lchA was 2130.9 ± 23.3 mAU×s,increasing 70.53%compared to the wild-type.Moreover,we knockout PlchA to block both Lichenysin and ComS synthesis,get an engineering strain WX-02 APlchA;and knockout the third open reading frame of lchA(IchAC)to only block Lichenysin synthesis,get an engineering strain WX-02 AlchAC.We haven't found Lichenysin in the broth of these two strains.Their broth produced less foams,cell growth haven't been disturbed yet.So that we achieve low-foam engineering strains.We detect the capacities of swarming and biofilm formantation of wild stain and three engineering strains.It is obvious that WX-02 P43lchA has greatly improved the capacities,and two deletion mutants weakened or lost the abilities compared to the wild strain.So it indicates that Lichenysin can promote microbial swarming and biofilm formation,which have great relationship with cell colonization and biological control?...