Artificial ribosomes for fully programmable synthesis of nonribosomal peptides
Provided herein, in some embodiments, are artificial ribosomes that synthesize nonribosomal peptides, polyketides, and fatty acids with full control over peptide sequence. Also provided herein are methods for programmed synthesis of nonribosomal peptides, polyketides, and fatty acids. In particular, provided herein are methods for scalable synthesis of a wide range of antibacterial, antifungal, antiviral, and anticancer compounds.
1. A molecular assembly line comprising:
(a) a synthetic RNA scaffold that comprises at least two aptamers, wherein each aptamer comprises a polypeptide binding site; and
(b) at least two nonribosomal peptide synthetase (NRPS) fusion proteins, each NRPS fusion protein comprising a NRPS module fused to a RNA-binding peptide that specifically binds to the polypeptide binding site of at least one aptamer within the synthetic RNA scaffold, wherein the synthetic RNA scaffold and the at least two NRPS fusion proteins form a complex.
2. The molecular assembly line of claim 1 , wherein at least one NRPS module is selected from licA1, licA2, licB1, licB3, licC1, tycB, tycC, lgrA, lgrB, lgrC, lgrD, grsB, srfA1, srfA2, and srfA3.
3. The molecular assembly line of claim 1 , wherein the synthetic RNA scaffold comprises tRNA analogs.
4. The molecular assembly line of claim 1 , wherein at least one RNA-binding peptide comprises an RNA-binding domain from a protein selected from Lambda N, P22N, RevN7D, HTLV-1-Rex, and BIV-Tat.
5. The molecular assembly line of claim 4 , wherein at least one RNA-binding peptide binds to an aptamer sequence within a tRNA analog.
6. A synthetic nucleic acid sequence encoding the molecular assembly line of claim 1 .
7. A biological cell comprising the synthetic nucleic acid sequence of claim 6 .
8. A method for assembling the molecular assembly line of claim 1 , the method comprising:
(a) providing the molecular assembly line to a cell-free expression system; and
(b) incubating the cell-free expression system containing the molecular assembly line under conditions wherein the NRPS fusion proteins are able to bind to the synthetic RNA scaffold.
9. The method of claim 8 , wherein at least one NRPS module is selected from licA1, licA2, licB1, licB3, licC1, tycB, tycC, lgrA, lgrB, lgrC, lgrD, grsB, srfA1, srfA2, and srfA3.
10. The method of claim 8 , wherein the synthetic RNA scaffold comprises tRNA analogs.
11. The method of claim 8 , wherein at least one RNA-binding peptide comprises an RNA-binding domain from a protein selected from Lambda N, P22N, RevN7D, HTLV-1-Rex, and BIV-Tat.
12. The method of claim 11 , wherein at least one RNA-binding peptide binds to an aptamer sequence within a tRNA analog.