Mechanistic studies of DepR in regulating FK228 biosynthesis in Chromobacterium violaceum no. 968

Abstract

DepR, a LysR-type transcriptional regulator encoded by the last gene of the putative min operon (orf21-20-19-depR) located at the downstream region of the anticancer agent FK228 biosynthetic gene cluster in Chromobacterium violaceum No. 968, positively regulates the biosynthesis of FK228. In this work, the mechanism underlining this positive regulation was probed by multiple approaches. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay (DIFA) identified a conserved 35-nt DNA segment in the orf21-orf22 intergenic region where the purified recombinant DepR binds to. Quantitative reverse transcription PCR (RT-qPCR) and green fluorescent protein (GFP) promoter probe assays established that transcription of phasin gene orf22 increases in the depR deletion mutant of C. violaceum (CvΔdepR) compared to the wild-type strain. FK228 production in the orf22-overexpressed strain C. violaceum was reduced compared with the wild-type strain. DepR has two conserved cysteine residues C199 and C208 presumed to form a disulfide bridge upon sensing oxidative stress. C199X point mutations that locked DepR in a reduced conformation decreased the DNA-binding affinity of DepR; T232A or R278A mutation also had a negative impact on DNA binding of DepR. Complementation of CvΔdepR with any of those versions of depR carrying a single codon mutation was not able to restore FK228 production to the level of wild-type strain. All evidences collectively suggested that DepR positively regulates the biosynthesis of FK228 through indirect metabolic networking.

Origin and bioactivities of thiosulfinated FK228

“…[8][9][10][11][12] FK228 has a bicyclic peptide framework produced biosynthetically by a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line with multiple accessory enzymes. [13][14][15][16][17] A signature disulfide linkage in FK228 is critical for structural stability when FK228 normally exists as a prodrug, and for bioactivity as a potent HDAC inhibitor when FK228 is reduced in vivo to afford two sulfhydryl (-SH) groups. Mechanistically, a -SH group on the long aliphatic chain of reduced FK228 (red-FK228) is able to chelate with zinc ion in the catalytic center of class I HDACs, thus inhibiting the enzyme activity, which leads to a cascade of epigenetic consequences.…”

Genomics-guided discovery of a new and significantly better source of anticancer natural drug FK228

“…The biosynthesis of FK228 in Chromobacterium violaceum No. 968 was extensively studied, revealing a thiotemplated, hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) assembly line with multiple accessory enzymes and a stress-inducible transcriptional regulator [ [9] , [10] , [11] , [12] , [13] ].

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