Cloning,Heterologous Expression And Characterization Of Lipoxamycin Biosynthetic Gene Cluster From Strptomyces Gelaticus Sge12

Streptomyces gelaticus Sge12,isolated from forest soil of Shengnongjia in Hubei Province,showed remarkable activities against pathogenic fungi such as rice blast fungus.Genome sequencing,chemical isolation and structure elucidation,cloning and heterologous expression of the target gene cluster,as well as biosynthesis study were conducted in this thesis in order to discover antifungal compounds produced by S.gelaticus Sge12 and to investigate their biosynthesis pathway.In the beginning,the genome of S.gelaticus Sge12 was sequenced,which revealed 37 secondary metabolite gene clusters according to analysis with anti SMASH.However no one of these gene clusters showed high similarity with known gene clusters for antifungal antibiotics,which might hinder the identification of the antifungal compound and cloning of their biosynthetic gene cluster.Nevertheless,it implied the novelty of the biosynthetic gene cluster for the unknown antifungal compound.A bacterial artificial chromosome(BAC)library of S.gelaticus Sge12 was constructed and screened via high throughput BAC Library EXpression and Analysis System(LEXAS)at the mean time by other in this laboratory,yielding three BAC clones(7G7 and others)able to produce antifungal activity in a heterologous expression host.In this thesis,the three BAC clones were confirmed belonging to one contig and sharing a ca.69 kb overlapping region through terminal sequencing and restricted digestion.Furthermore,heterologous expression of the three BAC clones in Streptomyces lividans SBT18 led to the production of antifungal activity with similar spectrum.These findings indicated that three BAC clones carried the same gene cluster producing the same bioactive compounds.Subsequently,S.lividans SBT18/7G7 was chosen to ferment and bioactive compounds were isolated using bioassay-guided method,leading to the identification of lipoxamycin and its analogs NE-M₂ and NE-B₁.Then,lipoxamycin gene cluster was located on a 21 kb region of7G7 by large-fragment deletion and heterologous expression.Eight structural genes lxn A?lxn B?lxn C?lxn D?lxn E?lxn F?lxn G and lxn H,as well as two positive regulatory genes lxn R1 and lxn R2 were determined to be essential for the production of lipoxamycin by single-gene in-frame deletion.A long-chain aliphatic amine intermediate was found to be accumulated in the lxn C mutant by metabolic profiling analysis based on LC-MS.Finally,the lipoxamycin biosynthetic pathway was proposed based on the results of gene deletion,bioinformatics analysis,and LC-MS analysis of the mutants.To sum up,the lipoxamycin biosynthetic gene cluster was successfully cloned,located and heterologously expressed in this work.The lipoxamycin biosynthesis pathway was also investigated through genetic manipulation.This study laid the foundation for the engineering of high-producing bacterial strains and the development of novel antifungals through combinatorial biosynthesis.