Discovery and overproduction of novel highly bioactive pamamycins through transcriptional engineering of the biosynthetic gene cluster

Background Pamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties. Results Herein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria. Conclusion By employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02231-x.


S. albus J1074 R2-p73down
S. albus J1074 strain with the R2 cluster and the semisynthetic promoter p73down This study  Legend: red -strains producing no pamamycin or with the yield significantly lower than in the control R2 carrying strain; green -strains with the pamamycin production on par or higher than that of S. albus J1074 P21pamW/R2.

Plasmids
pTOS derivative containing pamW gene under P21 promoter This work pUWLH pIJ101 replicon based replicative vector, HygR [8] pUWLHpamS pUWLH with cloned pamS gene This work pUWLHpamW pUWLH with cloned pamW gene This work R2 Derivative of pOJ436 containing the cluster for pamamycin biosynthesis [9] R2 mutant library R2 cosmids, containing a Hyg R cassette flanked by 2 random promoters between the pamF and pamA genes containing the upstream promoter of the R2/R2-67/R2-73 or R2-100 cosmids cloned in front of the gusA reporter gene This work pGUS-pR2Down/p67Down/p73Dow n/p100Down Derivative of pGUS containing the downstream promoter of the R2/R2-67/R2-73 or R2-100 cosmids cloned in front of the gusA reporter gene This work Table S2.Oligonucleotides used in this study amplification of the P67-Up promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaIR2-pamAp67-GU-R AAAAGGTACCAACGATTCCTCCGATAGCGA Primer for the amplification of the P67Down promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-pamFp73-GU-R AAAAGGTACCGCGACTTCCCTCCTTCACAACTCATCCTAACTCATCGCT AGCCGTTGGAGCCGGTACTAAATACTTGACATATCACTGT Primer for the amplification of the p73Up promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-pamAp73-GU-R AAAAGGTACCAACGATTCCTCCGATACCTC Primer for the amplification of the p73Down promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-pamFp100-GU-R AAAAGGTACCGCGACTTCCCTCCTTCATCTGCGATCCTAGCACGAAAC AGGGTATGACAGCCTGTGTTAAATACTTGACATATCACTGT Primer for the amplification of the P100Up promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-pamAp100-GU-R AAAAGGTACCAACGATTCCTCCGATAGAGG Primer for the amplification of the P100Down promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-pamA-GU-R AAAAGGTACCACGGGAAAGCTATCCGCCGG Primer for the amplification of the native downstream promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-PL-Nat-R AAAAGGTACCGAGACTCCCTGTGTTGCGTG Primer for the amplification of the native upstream promoter for the cloning into pGUS with restriction site SpeI R2-PL-Nat-F TATGTGAATCACAGTGATATGTCAAGTATTTGCCGGGAATTCGAGGGACA R2-PL-Nat-HygR-R GGTGGGCCGATGTCCCTCGAATTCCCGGCAAATACTTGACATATCACTGT Primer for the amplification of a Hyg R cassette for the cloning into pGUS with restriction site XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT R2-pamF-GU-R AAAAGGTACCGAGACTCCCTGTGTTGCGTG Primer for the amplification of the native upstream promoter and a Hyg R cassette for the cloning into pGUS with restriction sites SpeI and XbaI R2-GU-F-all AAAATCTAGATCAGGCGCCGGGGGCGGTGT Fig. S1.High resolution MS-chromatograms of S. albus J1074 R2-73.Pamamycin derivatives are indicated by their molecular weight.

Table S3 .
Schematic overview of pamamycin production by engineered R2 constructs expressed in 9

Table S5 .
Results of antibacterial tests of different pamamycins.

Table S6 .
Results of activity tests of pamamycins with different molecular weight against cell lines

Table S8 .
Results of activity tests of pamamycins with different molecular weight against Agrostis