Heterologous Expression And Function Study Of Three Gene Clusters In Halophilic Actinomycetes Amycolatopsis Aidingensis YIM96748

Actinomycetes are the main alternative source in the field of new drug research and development,and many secondary metabolites have been developed into antibiotics for clinical application.Due to objective environmental changes and the abuse of antibiotics,existing drugs are not enough to combat frequent outbreaks of epidemics,and new drug research and development is urgent and necessary.Special microbes grow in extreme environments,and genomes tend to be different from other habitat microbes in their continuous evolution and often have the ability to produce new natural products.Halophilic actinomycetes are a special microorganism,and there are few reports on its natural products and its research potential is high compared with other habitat actinomycetes.We have accumulated a large number of halophilic actinomycetes resources and also used traditional natural product screening approaches to find new compounds,but we have encountered many difficulties.We found that the fermentation work of halophilic actinomycetes mainly has the following difficulties compared with general actinomycetes:first,the growth of halophilic actinomycetes are extremely slow;second,the yield of fermentation products of halophilic actinomycetes are very low;third,the high concentration salt environment required for the growth of Halophilic actinomycetes will corrode fermentors and other equipment;fourth,the type and number distribution of gene clusters are extremely uneven.Therefore,compared with conventional actinomycete fermentation,these adverse characteristics of halophilic actinomycetes make its fermentation work extremely hard,which greatly limits the development and utilization of this type of resources.In the previous work,we used a combination of biosynthetic pathway-specific probes and real-time quantitative PCR to screen high-throughput strains with the potential to produce new compounds,some strains of halophilic actinomycetes were selected and their genome sequences were determined.Through genome mining,it was found that amycolatopsis aidingensis YIM 96748 had potential gene clusters to produce many kinds of new natural products.In order to solve the difficult problem of fermentation of halophilic actinomycetes,we adopted heterologous expression method.In this paper,the halophilic actinomycetes Amycolatopsis aidingensis YIM 96748 was studied.Three potential biosynthetic gene clusters 24,25 and 25-back of secondary metabolites were screened by genome sequencing and genomic mining clone library hybridization.The products may be lanthipeptide,polyenes compounds and type I polyketides compounds.Gene cluster 24 and Gene cluster 25 were successfully screened from YIM 96748 genomic library by colony in situ hybridization.After the clone was confirmed to be correct,it was transformed into E.Coli ET12567/PUZ8002,16 strains of heterologous expression engineering strain successfully constructed by conjugating and transferring into host strain Streptomyces albus J1074,Streptomyces coelicolor M145,Streptomyces lividans TK24 and Streptomyces lividans SBT18.According to the characteristics of the potential products of different gene clusters,different fermentation media were selected to carry out targeted fermentation,and the fermentation products were analyzed.Some of the strains showed new absorption peaks,but their fermentation stability remains to be further explored.This study preliminarily demonstrated that the biosynthetic gene cluster of halophilic actinomycetes YIM 96748 could be introduced into the engineered strain and may produce new products,and the successful construction of multiple heterologous expression engineered strains laid a foundation for following studies.