| Streptomyces roseoflavus Men-myco-93-63 is a biocontrol strain originally isolated from soil where potato scab was naturally declined.This strain and its fermentation broth have exhibited a good inhibitory effect against crop diseases such as cotton verticillium wilt,cucumber powdery mildew,tomato gray mold,and vegetable parasitic nematodes.Previously a group of pentaene macrolides mainly composed of two constituents roflamycoin and men-myco-A(referred to as R&M)was obtained from the fermentation broth of S.roseoflavus Men-myco-93-63.This group of antibiotics exhibited broad-spectrum antifungal activities in vitro,indicating their potential use in plant disease control.Roflamycoin is a polyene macrolide first reported in 1968.Its mechanism of action,chemical structure and de novo synthesis have been studied so far.In recent years,similar derivatives have also been reported.In this paper,based on the sequencing of the genome of S.roseoflavus Men-myco-93-63,the biosynthetic pathway of R&M produced by S.roseoflavus Men-myco-93-63 was identified via bioinformatic analysis and molecular biology,and the genes responsible for R&M biosynthesis in the gene cluster of R&M were studied as well.The main results are as follows:1.The biosynthesis process of R&M in S.roseoflavus men-myco-93-63 was analyzed by bioinformatics method,and its biosynthesis gene cluster location was identified.Through Pac Bio RS?sequencing,the genome of Men-myco-93-63 containing only one linear chromosome was obtained with a size of 8856609 base pairs,a GC content of 72% and 7692 predicted genes.Anti SMASH analysis suggested that it could produce 37 secondary metabolites,including polyketides(PKs),nonribosomal peptides(NRPs),ribosomally synthesized and post translational modified peptides(Ri PPs),terpenes,and other compounds;and a cluster 36,located in orf7375-7449,was predicted to be involved with R&M biosynthesis.Combining the the chemical structure and bioinformatic analysis,the biosynthesis process of RM was deduced: with isobutyryl-Co A(as final product was roflamycoin)or2-methylbutyryl-Co A(as final product was Men-myco-A)as the substrates,and malonyl-Co A or methylmalonyl-Co A as the extension units,a 17-step consecutive claisen condensation was processed to extend the polyketone chain.The full-length polyketide intermediate was then offloaded and cyclized to generate R&M.By analyzing the ?-ketoreductase(KR)domain of polyketide synthase(PKS),the stereostructure of R&M was considered to be consistent with the one reported.In addition,homology alignment analyses of PKS upstream and downstream genes found a number of transcriptional regulators,transporters and some genes with other functions,which were speculated to be related to R&M synthesis.These predicted genes related to R&M synthesis were named rfm.2.Twenty rfm gene inactivated mutants were obtained by gene knockout and five synthetic genes,two regulatory genes and one thioesterase gene were identified.Firstly,five PKS genes in the rfm gene cluster were blocked by homologous recombination and double exchanges,and all the mutants lost the ability of R&M synthesis.In addition,two amino acid residues in the catalytic center of ER2 domain mutated the conserved motif Lx Hxxx GGVGxx Axxx A to Ax Axxx GGVGxx Axxx A,generating R&M derivatives with C32-C33 being double bond.This confirmed that the five PKS genes were indeed responsible for the biosynthesis of R&M.Next,three genes(i.e.rfm W,rfm G,rfm J)involved in the synthesis of R&M were found by blocking or deleting 15 genes upstream and downstream of PKS genes:(1)The rfm W was the third gene upstream of PKS genes,which contained the DNA binding domain of HTH-XRE.The blocking of rfm W increased R&M production by 68.52% and 97.23%respectively.When using Streptomyces constitutive strong promoter erm Ep* for gene complement,it was found that the overexpression of rfm W slowed down the growth of S.roseflavus Men-myco-93-63.The appearance of aerial hyphae and spores was one day later than that of wild type strain.The production of antibiotics roflamycoin and men-myco-A decreased and the ratio of the two decreased from 5:2 to about 3:2.Meanwhile,q RT-PCR showed that rfm W did not significantly affect the expression of PKS genes.Therefore,rfm W was considered to be a global regulator,which was related to cell growth and differentiation,and amino acid metabolism simultaneously.(2)The rfm G gene was the first gene immediately downstream of the PKS genes.Blocking this gene reduced the yield of R&M by51.85% and might produce R&M derivatives with acetyl-Co A as the starting substrate(i.e.C-35 methyl substituted derivatives),indicating that rfm G was a type II thioesterase gene with error correction function.(3)The rfm J was the third gene downstream of PKS genes.It contained HTH-XRE DNA binding domain at N-terminal and MLTR-LBD(Mmyb-like transcription regulator ligand binding domain)domain at C-terminal.The deletion of this gene resulted in the loss of R&M synthesis ability,and accumulation of a series of R&M synthesis precursor intermediates at the position where the retention time was advanced.In this study,the biosynthetic gene cluster cloning of roflamycoin was reported for the first time via bioinformatics analysis combined with in vivo gene knockout experiment.These results may lay a foundation for the high yield and application of R&M in the future. |
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