Discovery Of Bioactive Natural Products Enabled By High Throughput Heterologous Expression And Biosynthesis Of A Novel Lantibiotic,Lexapeptide

An increasing number of microbial genomes are sequenced due to the rapid development of DNA sequencing technology.These microbial genomes are discovered to be rich sources of secondary metabolites'biosynthetic gene clusters(BGC)using bioinformatics analysis tools.In order to exploit these secondary metabolites'BGCs for the increasing demand of new drugs,a high throughput Bacterial artificial chromosome(BAC)Library EXpression and Analysis System(LEXAS)was demonstrated in this study.BAC library constructed in an Escherichia coli-Streptomyces shuttle vector was conjugated to the surrogate host Streptomyces lividans SBT5 in a high throughput way through tri-parental mating,using E.coli ET12567/p UB307 as the helper strain.Then high throughput micropore fermentation and bioassay test overlaid with indicator strains were followed to realize the antimicrobial screening of the exconjugants library for bioactive secondary metabolites.LEXAS was applied to screen the BAC library of Streptomyces rochei Sal35 in this study.Five BGCs governing the biosynthesis of borrelidin(Contig1,type I PKS),streptothricin(Contig2,peptidylnucleoside),two novel linear lipopeptides,8D1-1/2(Contig3,NRPS),a novel lantibiotic,lexapeptide(Contig4,lantibiotic),and a putative Class III lantibiotic(Contig5)were discovered from S.rochei Sal35.Thereinto,BGCs of the linear lipopeptides 8D1-1/2 and lexapeptide were‘silent'in S.rochei and uncovered by LEXAS for the first time.These results revealed that LEXAS could be employed to clone and activate the expression of cryptic BGCs encoding various kinds of bioactive natural products from Streptomyces efficiently and it is very suitable for functional genome mining in Streptomyces.Lexapeptide is a novel highly modified lantibiotic consisting of 38 amino acid residues.The post-translational modifications in lexapeptide include:N-terminal(N,N)-dimethyl phenyalanine;dehydroalanines at Ser2,Ser18 and Ser19 residues;dehydroaminobutyric acids at Thr4 and Thr12 residues;Me Lan formed between Cys22andAbu33residues;D-Ala28;and S-[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine(Avi Me Cys)formed between Cys38and Abu30 residues.These unique post-translational modifications qualify lexapetide with potent antimicrobial activity and stability.Using DNA sequencing and large DNA fragment deletion,a minimal plasmid p JLXM was obtained for lexapeptide biosynthesis,which bears 22,984 bp exogenous genomic DNA encoding 21 open reading frames.In order to elucidate the biosynthetic logic of lexapeptide,single gene deletions were made on p JLXM for metabolic analysis.Among those 21 genes,lxm A,lxm D,lxm W,lxm Y,lxm Z,lxm P1,lxm P2,lxm M,lxm J,lxm T,lxm R1,lxm R2,and lxm R3 were determined to be essential for lexapeptide biosynthesis.Thereinto,lxm A encodes the precursor peptide Lxm A;lxm D encodes a cysteine decarboxylase Lxm D probably catalyzing the decarboxylation of the C-terminal Cys in Lxm A;lxm M encodes a SAM-dependent methyltransferase Lxm M catalyzing the N-terminal(N,N)-dimethylation on phenylalanine;and lxm J encodes an F₄₂₀-dependent reductase Lxm J catalyzing the D-Ala installation.Lxm J shares no homology to either Lan J_A or Lan J_B protiens which were known to be involved in D-Ala and D-Abu installation in lantibiotics biosynthesis.Lxm J consists of a third family of protein for D-Ala installation in lantibiotics which was named Lan J_C in this study.The Lxm J_C-catalyzed stereospecific reduction reaction was reconstituted in vitro successfully using lexapeptide Z as substrate.Combining in vitro biochemical characterization and in vivo point mutations on lxm A,Lxm J_C was characterized to use F₄₂₀H₂ as cofactor and catalyze the reduction of Dha28 within the thioether-bridged ring structure in lexapeptide site specifically.Neither Dhb nor Dha present in the linear moiety of lexapeptide was observed to be reduced by Lxm J_C.In the lexapeptide BGC,there was no gene encoding Lan B or Lan C,Lan M,Lan KC,or Lan L homologous protein involved in the dehydration and cyclization reactions in lantibiotics biosynthesis.We proposed that Lxm WYZ should be involved in Ser and Thr dehydration and cyclo-addition of Cys to Dhb based on the bioinformatics and genetic analyses.According to the lantibiotics classification rules,lexapeptide was assigned to a Class V lantibiotic to discriminate from the Class I-IV lantibiotics.Further Blastp search in the NCBI database using Lxm WYZ proteins as queries revealed a number of BGCs for putative Class V lantibiotics.In summary,a novel Class V lantibiotic with lexapeptide representing the first member of the family was discovered and characterized in this study.The lexapeptide biosynthetic pathway discussed here sets the stage for future study of Class V lantibiotics.Meanwhile,the discovery of Class V lantibiotics enriches the family member of lantibiotics,and the novel enzymes encoded by Class V lantibiotic BGC also provide more synthetic biology tools for lantibiotics engineering.In particular,a series of lexapetide derivatives obtained in this study exhibit potent antimicrobial activity against Methicillin-resistance Staphylococcus aureus(MRSA)and Staphylococcus epidermis(MRSE),and would contribute significantly to the development of antimicrobial agents.