Functional Identification Of Gene Cluster For ε-polylysine Synthesis From Streptomyces Lavendulae X33

ε-poly-L-lysine(ε-PL)is a peptide polymer made of L-lysine.It has good water solubility,wide antibacterial spectrum and high thermal stability.and ε-PL is an efficient food preservative.In the preliminary work,Penicillium digitatum was used as the target to obtain a Streptomyces lavendulae X33 with strong antibacterial activity.Firstly we separate and purify the active substance,and the active substance was analyzed by TLC,and LC/MS,then we combined the genome sequencing results,It is speculated that the active substance produced by strain X33 fermentation may contain ε-polylysine as a component.In this study,Streptomyces lavendulae X33 was taken as the research object,the fermentation products were separated and identified,and the molecular mechanism of strain synthesis of ε-polylysine was studied.Methylene blue was used as an indicator,a transparent circle appeared around the colony of Streptomyces lavendulae X33 growing on solid fermentation medium,This phenomenon showed that the fermentation product of Streptomyces lavendulae X33 may contain alkaloids.The test results of Dragendorff reagent for Streptomyces lavendulae X33 fermentation broth also showed that the fermentation broth contained alkaloid components.The fermentation broth was separated and purified by D3 92 anion exchange resin and D152 cation exchange resin for detection.The purified product’s Dragendorff reagent test and bacteriostatic test showed positive results.The purified product’s Tricine-SDS-PAGE electrophoresis showed that the active product was small peptides The molecular weight is between 3.2-4.2 kDa,and the purified product is determined to be ε-PL by detection of HPLC.We sequenced the genome of Streptomyces lavendulae X33,refering the reportedε-polylysine synthetase gene cluster,we found a similar ε-polylysine synthetase gene cluster in the strain X33 genome by bioinformatics analysis.The gene cluster is composed of an ε-polylysine synthase encoding gene(pls)and a putative ε-polylysine degrading enzyme encoding gene(pld).The pls gene is 3996 bp and encodes a non-ribosomal peptide synthetase(NRPS)with a length of 1331 amino acids.Its molecular weight is 139.49 kDa and its isoelectric point is 7.3.The primers were designed to amplify the full length of pls gene withThe total DNA of strain X33 as a template,and the cloning vector was constructed for heterologous expression.It was found that pls could not be directly expressed in E.coli,and it is heterologously expressed in Streptomyces lividansTK24 alone.Amplifier with upstream 200 bp and pls were amplified together and constructed into shuttle integrated plasmid PIB139.After introducing Streptomyces lividans TK24 by conjugation transfer,the recombinant strain Streptomyces lividans TK24-pls fermentation product also had antibacterial activity.Column chromatography,Dragendorff reagent detection and HPLC detection of the active substance confirmed that it was consistent withε-PL,indicating that the pls gene was expressed in S.lividans TK24.PCR amplification of the presumed ε-polylysine degrading enzyme coding gene(pld)located downstream of the pls gene of strain lavendulae X33 to construct a heterologous expression vector pMAL-c5x-pld,and the recombinant vector was introduced into E.coli BL21(DE3).After IPTG induction,heterologous expression is achieved.Thepld is 1347 bp,and pldencodes 448 amino acids,and its molecular weight of approximately is 49.5 kDa.However,when ε-PL was as the substrate,it was found that the recombinant rPLD enzyme could not degrade the substrate.Amino acid sequence analysis showed that PLD belongs to the M1 group metallopeptidase.L-Leucine-4-nitroaniline(L-Leu-pNA)was used as the substrate to determine the enzymatic properties of the recombinant rPLD enzyme.The optimal reaction conditions for the recombinant rPLD enzyme were 40℃ pH 7.0,The enzyme activity was 32.91 U/mg,when the temperature was lower than 40℃,and the pH was between 6.0-8.0,the recombinant rPLD enzyme was relatively stable,and the amount of p-nitroaniline produced tended to be stable after 3 h.