Genome Mining Of Lanthipeptides From Microcystis Aeruginosa And Streptomyces Yunnanensis

Lanthipeptides are one superfamily of ribosome synthesis and post-translational modification peptides(RiPPs),which have a variety of biological activities,including antibacterial,antiviral and antifungal activities.Their characteristic structure is(methyl)lanthionine(Lan/MeLan).RiPPs follow the same biosynthetic logic:the precursor peptide consisting of the leader peptide and core peptide is gene-encoded and ribosomally produced,then post-translational modification(PTM)enzymes recognize the leader peptide,modifying on the core peptide,in the end,the leader peptide is excised by protease to obtain the mature RiPPs.Lanthipeptides are divided into five classes according to the different PTM enzymes that form lanthionine.The research focuses on the genome mining,engineering and biosynthesis of the class Ⅱ and class Ⅲ lanthipeptide.The class Ⅱ lanthipeptide synthetases,LanMs,are bifunctional enzymes consisting of N-terminal dehydration domain and C-terminal cyclization domain.The N-terminal domain activates the dehydration of Ser/Thr to Dha/Dhb,and the C-terminal domain catalyzes the attack of sulfhydryl of Cys to Dha/Dhb forming Lan/MeLan.The representative enzymes,ProcM and SyncM,recognize a wide variety of substrates,of which the leader peptide is relatively conservative,while the CP is more diverse.A gene cluster mal was identified from Microcystis aeruginosa NIES-88 via genome mining,encoding two precursors(MalAl and MalA2),a classⅡ lanthipeptide synthetase MalM,indicating that MalM may have substrate promiscuity.Through coexpression,artificial combination of precursor peptide,mass spectrometry and other methods,we identified 5 groups of related compounds 1-5.Further structure analysis and comparison,we found that MalM was a new class Ⅱlanthipeptide synthetase:even though MalM and ProcM share the characteristic three zinc-binding cysteines,they have disparate fashions for dehydration and cyclization on same cores fused to their respective cognate leaders.These data suggest that the PTM reactions catalyzed by MalM are affected by its regioselectivity rather than the sequence of core peptide.In bioinformatics analysis,we compared the sequences of LanM enzymes with potential substrate promiscusity,as well as their cognate precursor peptides,through constructing a sequence similarity network of LanMs and Maximum-likelihood tree of precursors,which provides evidences for the co-evolution feature of cognate precursor peptides and LanMs.These biochemical and bioinformatics results will facilitate lanthipeptide engineering for structural diversity by leader replacement.We found a gene cluster syc in Streptomyces yunnanensis CGMCC 4.3555 by genome mining,which contained sycKC,sycA and S9 protease genes sycP,encoding a putative class Ⅲ lanthipeptide.There are two genes encoding primary metabolism enzymes lkb downstream:glycerol kinase SycGK and alcohol dehydrogenase SycDH.The DNA fragment of syc-fl containing sycA,sycKC and sycP genes were heterologous expressed in model Streptomyces.M15,PTM and SSY medium were used to produce compounds S1-S4 with three-water-molecule loss.Compounds S1-S4 were identified as homologues with only difference at the N-terminal overhangs,which was proposed to be due to the prolyl oligopeptidase of SycP,specifically removing the leader at Pro—8,then the amino acid residues at the N-terminal were removed one by one by proteases with aminopeptidase activity outside the cluster.The heterologous expression of syc-f2 fragment containing the additional glycerol kinase SycGK and alcohol dehydrogenase SycGK was carried out using same procedures.A variety of Streptomyces hosts and 6 culture media were screened,but no additional product was identified,suggesting that SycGK and SycDH are not involved in biosynthesis of target lanthipetide.The fermentation of wild type S.yunnanensis CGMCC 4.3555 also produce compounds S3 and S4,further supporting the above analysis.As the yield from wild type strain is better,we determine to isolate compounds S3 and S4 using wild type strain.The 10L cultures were absorbed and fragmentated by macroporous resin chromatography,and then S3 and S4 were separated and purified by reversed C18 silica gel and Sephadex LH20 chromatography.Based on the tandam mass and NMR analysis,compound S3 and S4 were determined to contain a labionin ring(Dhal,Dha5,and Cys13)and a Dhb residue.This is in consistent with the result of the reaction with βME yielding a single βME adduct.In addition,this thesis summarizes genome mining tools and engineered strategies of RiPPs previously reported.The methods of engineering for RiPPs with diverse structures and excellent bioactivity include construction of chimeric precursors,in vitro translation system,incorporation of non-canonical amino acids,changes on growth conditions,rational design of RiPPs biosynthetic enzymes,and RiPP library generation by DNA synthesis techniques for degenerate codons and peptide surface display.