Study On The Biosynthesis Process Of Polyketides Derived From Soil Microorganisms

Natural products of microbial origin have important biological activities and play an important role in the pharmaceutical,agricultural,chemical and other industries.Among them,polyketides are a large class of natural products with important biological activities,including the anticancer drugs doxorubicin and mithramycin,and the antibiotic tetracycline.Polyketides compounds are usually synthesized by polyketide synthases(PKS)catalyzed polyketide skeleton,and then through a series of modification reactions to form polyketide compounds with biological activity,such as oxidation-reduction,group transfer,cyclization reactions,etc.The biological activity of polyketide compounds is closely related to their structure,and it is of great significance to study their biosynthetic processes.By studying the diversity of the polyketide biosynthesis process,the biosynthetic pathway of the polyketide compound can be manipulated or redesigned to generate new drug candidates,and it can provide a reference for the biosynthesis process of other polyketide compounds.The cosZF484 is a single clone previously identified by the research group that contains a type Ⅱ polyketide gene cluster.It was transferred into Streptomyces albus J1074 and specific peaks were produced by fermentation.Due to the complexity of the structure,only part of the structure was determined.Based on this part of the structure,it was presumed to be a new type Ⅱ polyketide compound.In this study,the PCR-targeting method was used to knock out the functional genes on the type Ⅱ polyketide biosynthetic gene cluster of cosZF484 discovered earlier by the research group,aiming to study the biosynthesis process of its the fermentation specific products and help analyze the structure of the specific products.The cosZF484 contained two cytochrome P450 genes on the type Ⅱ polyketide biosynthesis gene cluster,which generally played a role of post-modification of the compound structure during the biosynthesis process,but it is not common in the type Ⅱ polyketide biosynthesis gene cluster.In order to explore the biological function of P450 gene,we first knocked out two P450 genes and constructed the mutant 484-Δorf8 and 484-Δorf10,respectively.The mutants were heterologously expressed in S.albus J1074.Compared with the fermentation products of cosZF484 before knock out,the HPLC profiles of two P450 mutant fermentation broth revealed new specific peaks.After a large number of fermentation and isolation,the structure is resolved by high-resolution mass spectrometry and NMR.484-Δorf8 fermentation produced a new specific peak 484-Δorf8-1(m/z 269.0815[M+H]+),the NMR data is being analyzed.484-Δorf10 fermentation produced two new specific peaks,484Δorf10-1(m/z 273.1127[M+H]+)and 484-Δorf10-2(m/z 255.1024[M+H]+),and the same structure was not found in the SCI finder database based on the related NMR data.For other gene knockout experiments on gene clusters,new specific peaks were also found in the fermentation products of 484-Δorf11(oxymethyltransferase)and 484-Δorf13(hypothetical protein),which are being accumulated by fermentation.The cosZF14 is a single clone previously identified by the research group that contains a type I polyketide gene cluster.It was heterologously expressed in S.albus J1074 by conjugation.A red conjugant was found on the conjugation plate,and three specific peaks were found in its fermentation broth by HPLC analysis.Through fermentation accumulation,separation and purification,and then using NMR and single crystal diffraction to determine the structure of one of specific peaks,there was not found the same structure of microbial source in SCI finder database.We named it salumycin.Through DPPH free radical scavenging experiment,we found that salumycin has moderate antioxidant activity.The result of early PCR verification of the genome of red conjugant indicated that the gene cluster in cosZF14 was not inserted into the genome of S.albus J1074.In order to study the origin of specific peaks,it is necessary to perform genome sequencing on the red conjugant to confirm whether the gene cluster in cosZF14 is inserted into the genome of S.albus J1074.To sum up,this study based on PCR-targeting system to knock out the functional genes on the type II polyketide biosynthetic gene cluster in cosZF484.The mutants were transferred to S.albus J1074 by conjugation.Specific products were obtained through a large amount fermentation in the medium and a series of separation.The specific peaks in fermentation products of mutant 484-Δorf10 have been analyzed by NMR.The specific peak in fermentation products of mutant 484-Δorf8 has been separated,purified and is being analyzed by NMR.The fermentation products of the other two mutants(484-Δorf11,484Δorf13)were being accumulated.In addition,a new compound salumycin with antioxidant activity was obtained from the fermentation products of the mutant produced by cosZF14 heterologous expression in S.albus J1074.