Development Of Microbial Natural Products Based On Enabling Technology Of Synthetic Biology

Microorganisms are the so-called“cell factory”for the biosynthesis of natural products,which can use their endogenous secondary metabolic network to assemble small molecular substrates such as carboxylic acids,amino acids and sugars into complex and diverse mature products.The relative processes involve the precise control and synergy of a large number of proteins.In microorganisms,the coding genes of these proteins are always arranged in clusters,which are named as biosynthetic gene clusters（BGCs）.Nowadays,the rapid development of the enabling technology of synthetic biology promotes the construction of artificial microorganisms as ideal“cell factory”.Through design and engineering of microorganisms to produce specific natural products,the transformation of the biosynthetic pathways of microbial natural products mainly involves the cloning,editing and expression of BGCs.This paper focuses on the production,transformation and discovery of microbial natural products to achieve efficient synthesis of active natural products,structural derivation of new natural products and directional mining of unknown natural products by using and developing advanced synthetic biology enabling technologies,such as Saccharomyces cerevisiae transformation-associated recombination（TAR）,CRISPR/Cas9 editing and Red/ET recombination.Lincomycin is a broad-spectrum antibiotic,which has a good inhibitory effect on many pathogenic bacteria.Our previous studies have shown that knockout of the lmb V gene can obtain a lincomycin intermediate containing ergothioneine.In order to prepare lincomycin and its intermediates efficiently,we used CRISPR/Cas9-TAR coupling technology to capture the lincomycin BGC,and knocked out the lmb V gene.The resultant gene clusters were subjected to heterologous expression in Streptomyces lividans TK24 and Streptomyces coelicolor M145.However,fermentation of the recombinant strains failed to produce lincomycin or its intermediate.It was found through RT-PCR that the lincomycin biosynthetic gene cluster was not activated in Streptomyces lividans TK24.Therefore,attempts to overexpress the positive regulatory gene lmb U are supposed to activate the gene clusters in heterologous hosts.Sch40832 is a bicyclic thiopeptide containing the core ring of dihydroimidazo piperidine,which has antitumor activity.At present,little is known about the biosynthetic mechanism of Sch40832.In this study,the CRISPR/Cas9-TAR coupling technology was used to capture the Sch40832 BGC for a series of follow-up genetic operations to further study the biosynthetic pathway of Sch40832 and the functional study of related genes.Thioviridamides,showing good apoptosis inducing activity,are a class of ribosomally synthesized and posttranslationally modified peptide containing characteristic thioamide bonds and a 2-aminovinyl-cysteine ring.At present,the research on the post-translational modification of thioviridamides is still limited.The mutant strains that knocked out the tva G _S-87and tva J_S-87genes were fermented,and the biosynthetic pathway of TVA-YJ was inferred through LC-MS detection.However,the compound production of the mutant strains was too low to identify their chemical structure.In this study,the newly constructed p JTU2554-?tva G_S-87and p JTU2554-?tva J_S-87were successfully introduced into the?tva G_S-87and?tva J_S-87mutant strains by conjugal transfer.The resultant recombinant strains can accumulate more target compounds,which are conducive to the subsequent research on the biosynthetic mechanism of the structural modification of thioviridamides.