Reconstruction And Heterologous Expression Of The Biosynthetic Pathway Of Anti-infective Teixobactin

In recent years,widespread use and even abuse of antibiotics has led to serious bacterial drug-resistance.Infections caused by drug-resistant pathogenic bacteria have become a thorny problem worldwide and endangered human health.Teixobactin,a depsipeptide antibiotic discovered in an uncultured soil microorganism,possesses a novel and unique mode of action against bacterial growth by binding to the highly conserved motifs of both lipid II(precursor of peptidoglycan)and lipid ?(precursor of cell wall teichoic acid)to inhibit cell wall synthesis.Such a particular mechanism makes it to be a leading compound with great potential against drug-resistant pathogens.It has been reported to kill a variety of drug-resistant pathogens,including clinically resistant pathogenic bacteria such as methicillin-resistant Staphylococcus aureus(MRSA)and Mycobaterium tuberculosis.Since the discovery of teixobactin in 2015,its biological activity,mode of action,and structure-activity relationship(SAR)have been gradually studied.Though its total chemical synthesis has also been achieved,difficulties in chemical synthesis of a rare unnatural amino acid L-allo-enduracididine(L-allo-End)and the thirteen-membered ring greatly limit the yield improvement via chemical synthesis.Therefore,the biosynthesis of teixobactin in culturable microorganisms is alternative and critical for the future drug development.Based on the cluster sequence released in NCBI,using gene chemical synthesis,Red/ET recombineering and ExoCET recombineering,we carried out segmental synthesis of genes,assembly,genetic engineering and finally reconstructed the teixobactin artificial biosynthetic gene cluster(about 52 kb).Next,we obtained a serial of expression plasmids harboring this artificial biosynthetic gene cluster of teixobactin,followed by introducing it into the appropriate expression hosts.We measured the liquid cultures of the resultant recombinants and detected obvious accumulation of a series of new compounds,which showed antibacterial activity against Bacillus subtilis.Then,we isolated and purified these compounds and carried out analysis of their chemical structures by high resolution mass spectrometry-ultra-high pressure liquid chromatography(HPLC-HR-ESIMS)and nuclear magnetic resonance(NMR).Because the hosts and the resultant cluster contain no the gene cluster for its rare precursor-the unnatural amino acid L-allo-End,firstly we used the acid-degraded enduracidin containing L-End and D-allo-End to feed the resultant recombinants as above,but failed to detect the production of teixobactin or related derivatives.Next,we reconstructed gene clusters for the biosynthesis of the important precursor-End,with gene chemical synthesis and DNA assembly using the sequence online.Then,we stitched the gene clusters of the teixobactin and End to obtain the whole teixobactin-End gene clusters.These heterologous expression in the host S.albus J1074 led to accumulation of some new compounds with larger molecular weight than those compounds as above.In a conclusion,we reconstructed teixobactin gene clusters originated from an unculturable microorganism and functionally expressed in culturable heterologous host,and obtained a serial of new compounds with obvious antibacterial bioactivity.Though our trial to offer its unnatural precursor L-allo-End has failed,our data still will be instructive to promote the development of new anti-infective peptide drugs,and contribute to the innovative development of a new generation of antibiotics against drug resistance.