Construction and heterologous expression of the di-AFN A_(1) biosynthetic gene cluster in Streptomyces model strains

Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fold higher biological activities,compared with the monomer.Unfortunately,the yield of di-AFN A_(1) is very low(0.09±0.03 mg·L^(−1))in the engineered strain Streptomyces alboflavus 313_hmtS(S.albo/313_hmtS),which is not friendly to be genetically engineered for titer improvement of di-AFN A_(1) production.In this study,we constructed a biosynthetic gene cluster for di-AFN A_(1) and increased its production through heterologous expression.During the collection of di-AFN A_(1) biosynthetic genes,the afn genes were located at three sites of S.alboflavus 313 genome.The di-AFN A_(1) biosynthetic gene cluster(BGC)was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24,which produced di-AFN A_(1) at a titer of 0.43±0.01 mg·L^(−1).To further increase the yield of di-AFN A_(1),the di-AFN A_(1) BGC was multiplied and split to mimic the natural afn biosynthetic genes,and the production of di-AFN A_(1) increased to 0.62±0.11 mg·L^(−1) in S.lividans TK24 by the later strategy.Finally,different Streptomyces hosts were tested and the titer of di-AFN A_(1) increased to 0.81±0.17 mg·L^(−1),about 8.0-fold higher than that in S.albo/313_hmtS.Successful heterologous expression of di-AFN A_(1) with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.