Biological Modification Of Thiopeptide Antibiotic Nosiheptide Directed By Specific Structural Unit

Thiopeptide antibiotics are classes of natural products of polyazolyl peptides,which are rich in sulfur and highly modified.They are produced by secondary metabolism of microorganisms and have good biological activity.Developing thiopeptides into clinic is currently a challenge partly due to their structural complexity and poor aqueous solubility.Therefore,on the basis of understanding the mechanism of its biosynthesis,the method of obtaining thiopeptide analogues through reasonable bioengineering has become the focus of biologists.Nosiheptide?NOS?and thiostrepton?TSR?,as the star molecules of the thiopeptide family,have good physiological activities such as antibacterial,antimalarial and anticancer,and thus they have been widely studied and reported.The NOS side ring structure contains an indolic acid?4-methyl-2-indolyl acid,MIA?unit,and the TSR side ring contains a quinaldic acid?QA?unit,both of which are derived from L-tryptophan and independent of the precursor peptide..According to previous reports,the modification of QA units can obtain thiopeptide analogues with increased physiological activity.Therefore,in this paper,the mutants whose MIA unit synthesis is blocked are taken as the research object to obtain NOS analogues with improved physiological activity by using mutational biosynthesis.First,we chemically synthesized 9 kinds of MIA analogues by a simple and efficient method.In order to obtain more diverse MIA analogues,we purchased 14kinds of MIA derivatives.The mutant strain?nosL with nosL gene deletion responsible for MIA unit formation was used as a basal bacterium to ferment MIA derivatives.In all 22 fermentation broths,10 mature bicyclic NOS analogs were detected:5'-F-NOS,6'-F-NOS,5'-Cl-NOS,6'-Cl-NOS,5'-CH₃-NOS,9'-CH₃-NOS,O-NOS,S-NOS-1,S-NOS-2,S-NOS-3;9 monocyclic NOS analogs:5'-F-NOSint,6'-F-NOSint,6'-Cl-NOSint,6,7-2Cl-NOSint,7'-F-NOSint,7'-Cl-NOSint,5,6-2-F-NOSint,5'-F-6'-Cl-NOSint,5'-O-NOSint.When the hydrogen in MIA is replaced by a larger group:-NO₂,-CF₃,-Ph,no NOS-related compounds can be obtained.It can be seen from the feeding results that the volume and site of the substituent in MIA are the key factors affecting the upload of the substituent to the macrocyclic skeleton.The presence of fluorine atoms in the thiopeptide can usually improve its physiological activity.Among the NOS analogues we obtained,6'-F-MIA can be better tolerated by the macrocyclic framework,and two NOS analogues were obtained at the same time.According to the unique contribution of the MIA unit to the antibacterial activity of NOS,we hope to improve the NOS activity by changing the MIA structure.Therefore,in this experiment,the mutant strain?nosL fermentation broth was fed with a large amount of 6'-F-MIA and the corresponding NOS analogs were isolated.In the early stage of the experiment,we used the traditional fermentation method in the laboratory:MIA analogues were fed when primary fermentation strains were transferred to secondary fermentation,but only NOS analogues at mass spectrometry level were obtained.In order to improve the yield of NOS analogs,we explored the fermentation conditions,and finally determined that theprimary fermentation was used for feeding.This condition shortened the fermentation time to half of the original,the yield reached the level of HPLC analysis,and greatly improved the fermentation efficiency.This condition shortened the fermentation time to half of the original,the yield reached the level of HPLC analysis,and greatly improved the fermentation efficiency.After the large-scale fermentation and feeding of the?nosL mutant,the bicyclic NOS analog 6'-F-NOS and the monocyclic NOS intermediate 6'-F-NOSint were separated and purified from the bacterial cells by silica gel column chromatography and semi-preparative HPLC separation methods,and their structures were analyzed and identified by high-resolution mass spectrometry and NMR.In the antibacterial activity test,we selected methicillin-resistant staphylococcus aureus?MRSA ATCC43300?and vancomycin-resistant enterococcus?VRE ATCC29212,VRE ATCC51299,VRE ATCC51559?as subjects to study the activity of6'-F-NOS and 6'-F-NOSint.Unfortunately,the antibacterial activity of compound 6'-F-NOS is slightly lower than that of NOS,and the antibacterial activity of 6'-F-NOSint is completely lost.It can be seen from the above results that the modification of MIA unit can not only affect the antibacterial activity of NOS but also its water-solubility,which makes it difficult to develop new NOS analogs with improved physiological activity.Sidering-opening increases the degree of freedom of 6'-fluoroNOSint,while affects the binding of 6'-fluoro-NOSint to the target at the same time.In summary,we fed 6'-F-MIA to the mutant strain?nosL whose MIA synthesis was blocked,and obtained mature bicyclic 6'-F-NOS and single-ring 6'-F-NOSint via mutational biosynthesis.This work gives us a further understanding of the biosynthesis mechanism of NOS and lays the foundation for the biosynthesis of NOS analogs.