Construction and heterologous expression of the di-AFN A 1 biosynthetic gene cluster in Streptomyces model strains.

Natural cyclohexapeptide AFN A fromStreptomyces alboflavus 313 has moderate antibacterial and antitumor activities. An artificial designed AFN A homodimer, di-AFN A, is an antibiotic exhibiting 10 to 150 fold higher biological activities, compared with the monomer. Unfortunately, the yield of di-AFN A is very low (0.09 ± 0.03 mg·L) 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 production. In this study, we constructed a biosynthetic gene cluster for di-AFN A and increased its production through heterologous expression. During the collection of di-AFN A biosynthetic genes, the afn genes were located at three sites of S. alboflavus 313 genome. The di-AFN A biosynthetic gene cluster (BGC) was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24, which produced di-AFN A at a titer of 0.43 ± 0.01 mg·L. To further increase the yield of di-AFN A, the di-AFN A BGC was multiplied and split to mimic the natural afn biosynthetic genes, and the production of di-AFN A increased to 0.62 ± 0.11 mg·L in S. lividans TK24 by the later strategy. Finally, different Streptomyces hosts were tested and the titer of di-AFN A increased to 0.81 ± 0.17 mg·L, about 8.0-fold higher than that in S. albo/313_hmtS. Successful heterologous expression of di-AFN A with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.