Combinatorial libraries of synthetic and natural products are an important
source of molecular information for the interrogation of biological targets
. Methods for the intracellular production of libraries of small, stable mo
lecules would be a valuable addition to existing library technologies by co
mbining the discovery potential inherent in small molecules with the large
library sizes that can be realized by intracellular methods. We have explor
ed the use of split inteins (internal proteins) for the intracellular catal
ysis of peptide backbone cyclization as a method for generating proteins an
d small peptides that are stabilized against cellular catabolism. The DnaE
split intein from Synechocystis sp. PCC6803 was used to cyclize the Escheri
chia coil enzyme dihydrofolate reductase and to produce the cyclic, eight-a
mino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydr
ofolate reductase displayed improved in vitro thermostability, and pseudost
ellarin F production was readily apparent in vivo through its inhibition of
melanin production catalyzed by recombinant Streptomyces antibioticus tyro
sinase. The ability to generate and screen for backbone cyclic products in
vivo is an important milestone toward the goal of generating intracellular
cyclic peptide and protein libraries.