Abstract:
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.