Research And Construction Of Erythromycin Combinatorial Biosynthesis System

With unique chemical structure and biosynthesis mechanism,polyketides provide unprecedented opportunities for people to transform their components by the way of Combinatorial Biosynthesis to obtain a library of natural compound analogs.At present,the Combinatorial Biosynthesis based on the biosynthesis pathway of polyketone compounds has been well developed.Erythromycin is one of the broad-spectrum macrolide antibiotics which is widely used clinically for the treatment of Gram-positive and some Gram-negative bacterial infections.Due to its characteristics of good drugability and hypoallergenicity,it plays a very important role in the clinical antibacterial drugs and also shows the great potential in the development of new drugs.And what is fascinating is that there are many opportunities for people to transform the structure of the final compound of erythromycin by manipulating the biosynthetic pathway of polyketone compounds.Therefore,based on the erythromycin,we could obtain more novel erythromycin-like compounds with new structure and better drug activity by the method of Combinatorial Biosynthesis technology based on the transformation of biosynthetic pathway.Some gene editing systems have been successfully applied to actinomycetes(such as CRISPR / Cas9).In addition,erythromycin 14-membered macrocyclic skeleton 6-deoxyerythromycin lactone(6-d EB)as the most important intermediate in the synthesis of erythromycin,the more we study its synthesis mechanism,the easier we can enrich erythromycin skeleton types.Currently,scientists are also studying on heterologous biosynthesis of erythromycin to find more manageable and suitable hosts for erythromycin expression.There is a case that scientists have successed in establishing E.coli as a viable option for the heterologousproduction of erythromycin A.In order to establish an efficient combinatorial biosynthesis system,in this work we first tried to construct an erythromycin biosynthesis basics in Saccharopolyspora erythraea HL3168 using the CRISPR / Cas9 editing system p MWCas9.We taked S.erythraea HL3168 as the research object and knocked out the ery AI and ery AII genes.Although we successfully introduced the right plasmid into the erythromycin-producing strain,the mutant without ery AI and ery AII was not found.Secondly,the cosmid p DEBS-Ant E containing a complete 6-d EB synthetic gene cluster was modified to successfully construct a 6-d EB heterologous expression system in Mycobacterium smegmatis and it was expected to produce 6-d EB.However,6-deoxyerythromycin lactone was not detected in the fermentation broth.This probably because the expression of the recombinant plasmid was disturbed or the fermentation conditions may not be satisfied to catalyze the synthesis of the target product.Finally,in order to obtain a rapid analysis system for erythromycin,we tried to establish a biosynthesis system of erythromycin carbon skeleton in vitro.We expressed and reconstituted the DEBS1 as a dissociated complex of three proteins — a loading didomain LDD,module 1 M1 and module 2 M2,and finally obtain a protein complex with DEBS2 and DEBS3.When all five proteins were mixed at concentrations of 2 u M each,along with the requisite propionyl-Co A,methylmalonyl-Co A,and NADPH substrates,we carried out the6-d EB synthesis reaction similiar to in bacteria.However,we did not detect 6-d EB in the reaction solution.This probably because that reaction conditions affected the tertiary structure of the DEBS proteins,leading to a reduction in polyketide synthase activity.In this work,the erythromycin efficient biosynthesis system was researched and built from two ways—— in vivo and in vitro.Although no good results have been obtained,it can provide guidance for the subsequent establishment of an efficient synthesis system.