Cloning and heterologous expression of the new biosynthetic gene cluster of noncaromin from Nonomuraea candida HMC10T

Abstract:

[Objective] To obtain the new lasso peptide noncaromin, the biosynthetic gene cluster of noncaromin (nonc-BGC) from Nonomuraea candida HMC10^T was cloned and the heterologous expression in actinomycete classis strains as well as the antibacterial activity were explored. [Methods] The new nonc-BGC was initially defined by analyzing the whole genome sequence of N.candida HMC10^Twith antiSMASH. Then, complete nonc-BGC was cloned by ExoCET method from the genomic DNA of N.candida HMC10^T to obtain the recombinant plasmid pJQK652. The λ-Red recombination system was used to construct the integrative plasmid pJQK653, which, through conjugative manipulation, was introduced into Streptomyces albus J1074, S.lividans LJ1018, S.lividans 1326, S.coelicolor M1252, S.coelicolor M1452 and Saccharopolyspora erythraea LJ161 for heterologous expression. Subsequently target noncaromin was obtained by fermentation and purification. Finally, the chemical structure of noncaromin was determined by QTOF-ESI-MS², and its antibacterial activity was evaluated as well. [Results] In this study, complete nonc-BGC was cloned by EcoCET method, and its heterogeneous expression in six different actinomycete hosts were performed separately. Furthermore, we characterized the chemical structure of noncaromin and determined its negligible inhibition activity against Bacillus subtilis 168.[Conclusion] On the basis of the cloning of complete nonc-BGC from N.candida HMC10^T, the heterogeneous expression of nonc-BGC in six different actinomycete hosts was performed. As a result, a new noncaromin was discovered, with negligible inhibition activity against B.subtilis 168. These results provided reference for the discovery of new compounds in strain N.candida HMC10^T and other actinomycetes.