Agarose-embedded chromosomal DNA combines with ExoCET technology to capture the biosynthetic gene clusters of natural products in Actinobacteria

Abstract:

[Objective] To develop a method of cloning biosynthetic gene clusters of natural products (NP-BGCs) by combining agarose-embedded chromosomal DNA strategy with exonuclease combined with RecET recombination (ExoCET) technology and transform the cloned gene clusters into chassis cells for the expression of target NP-BGCs in heterologous hosts.[Methods] Firstly, the chromosomal DNA of the targeted strain was prepared by agarose-embedded plugs with the low-melting-temperature agarose and digested with the restriction enzymes to yield the linear DNA sample. Then, the linear target BGC was captured by the linear vector of p15A through the ExoCET technology. The desired integrative and conjugative elements were introduced into the BGC-containing plasmid through PCR-targeting approach. Subsequently, the final modified plasmid was introduced into Streptomyces coelicolor M1252 by intergeneric conjugation to yield the desired recombinant strains. Finally, the recombinant strains were fermented and analyzed for target compound production by UPLC-ESI-MS and the inhibitory activity against different indicator strains was detected. [Results] With this method, the BGC of lincomycin (lmb-BGC) and the BGCs of two ribosomal peptides (nioblantin, niob-BGC and nitblantin, nitb-BGC) were obtained from S.lincolnensis NRR2936 and Nonomuraea nitratireducens WYY166^T, respectively. Finally, the lmb-BGC was expressed in M1252 for production of lincomycin. [Conclusion] In this study, the lmb-BGC and two novel lanthipeptide BGCs were cloned by the agarose-embedded chromosomal DNA in combination with ExoCET technology. Then, the BGC-containing plasmids were modified for conjugations. The recombinant strains MJX01, MJX02, and MJX03 were obtained by conjugation with the strain M1252 host. The fermentation broth was extracted and analyzed by UPLC-ESI-MS and the anti-bacterial activity was detected. Finally, our results revealed that the lincomycin was successfully produced in the strain M1252 containing the lmb-BGC. This study lays the foundation for the discovery of new compounds through gene cluster cloning and heterologous expression in the chassis strain.