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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. [ABSTRACT FROM AUTHOR]

The presence of androgen receptor variant 7 (AR-V7) variants becomes a significant hallmark of castration-resistant prostate cancer (CRPC) relapsed from hormonal therapy and is associated with poor survival of CRPC patients because of lacking a ligand-binding domain. Currently, it still lacks an effective agent to target AR-V7 or AR-Vs in general. Here, we showed that a novel class of agents (thailanstatins, TSTs and spliceostatin A analogs) can significantly suppress the expression of AR-V7 mRNA and protein but in a less extent on the full-length AR expression. Mechanistically, TST-D is able to inhibit AR-V7 gene splicing by interfering the interaction between U2AF65 and SAP155 and preventing them from binding to polypyrimidine tract located between the branch point and the 3′ splice site. In vivo, TST-D exhibits a potent tumor inhibitory effect on human CRPC xenografts leading to cell apoptosis. The machinery associated with AR gene splicing in CRPC is a potential target for drugs. Based on their potency in the suppression of AR-V7 responsible for the growth/survival of CRPC, TSTs representing a new class of anti-AR-V agents warrant further development into clinical application. [ABSTRACT FROM AUTHOR]

Spiruchostatins A and B are members of the FK228-family of natural products with potent histone deacetylase inhibitory activities and antineoplastic activities. However, their production in the wild-type strain of Pseudomonas sp. Q71576 is low. To improve the yield, the spiruchostatin biosynthetic gene cluster ( spi) was first identified by rapid genome sequencing and characterized by genetic mutations. This spi gene cluster encodes a hybrid biosynthetic pathway similar to that encoded by the FK228 biosynthetic gene cluster ( dep) in Chromobacterium violaceum No. 968. Each gene cluster contains a pathway regulatory gene ( spiR vs. depR), but these two genes encode transcriptional activators of different classes. Overexpression of native spiR or heterologous depR in the wild-type strain of Pseudomonas sp. Q71576 resulted in 268 or 1,285 % increase of the combined titer of spiruchostatins A and B, respectively. RT-PCR analysis indicates that overexpression of heterologous depR upregulates the expression of native spiR. [ABSTRACT FROM AUTHOR]

Burkholderia species have emerged as a new source of diverse natural products. This mini-review covers all of the natural products discovered in recent years from Burkholderia sp. by genome-guided approaches-these refer to the use of bacterial genome sequence as an entry point for in silico structural prediction, wet lab experimental design, and execution. While reliable structural prediction based on cryptic biosynthetic gene cluster sequence was not always possible due to noncanonical domains and/or module organization of a deduced biosynthetic pathway, a molecular genetic method was often employed to detect or alter the expression level of the gene cluster to achieve an observable phenotype, which facilitated downstream natural product purification and identification. Those examples of natural product discovery from Burkholderia sp. provide practical guidance for future exploration of Gram-negative bacteria as a new source of natural products. [ABSTRACT FROM AUTHOR]

Acyldepsipeptides ( ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/ F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a β-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes. [ABSTRACT FROM AUTHOR]

Thailandepsin A is natural product of Burkholderia thailandensis E264 with potent histone deacetylase inhibitory activities and promising anticancer activities. The titer of thailandepsin A is very low (less than 10 mg/l) from limited empirical fermentation. To facilitate preclinical evaluations and potentially clinical development of thailandepsin A, systematic optimization and extractive fermentation of thailandepsin A from B. thailandensis E264 culture in flasks were investigated in this pilot study. The main fermentation parameters-28°C, pH 7.0, inoculum ratio 1% (v/v), incubation duration 60 h, medium volume 26%, shaking speed 170 rpm, and chloroform as extracting solvent-were determined by single factor experiments. Polyaromatic adsorbent resin Diaion HP-20, when added at a concentration of 4% (w/v), was most effective to reduce feedback inhibition of thailandepsin A and to significantly increase the titer of target product. Central composite design was used to further optimize the fermentation medium for B. thailandensis E264. The optimized medium contains glucose 17.89 g/l, tryptone 34.98 g/l, potassium phosphate 24.84 g/l, and sodium citrate 0.01 g/l, which resulted in a large increase of the titer of thailandepsin A to 236.7 mg/l. Finally kinetic models based on the modified logistic and Luedeking-Piret equations were developed, delivering a good description of temporal variations of biomass, product, and substrate in the fermentation process, which could be used as references for developing large-scale fermentation. [ABSTRACT FROM AUTHOR]

The article presents a comparative analysis of the large conformational change of the pseudomerohedrally twinned monoclinic structure of unfolded free nonactin upon the encapsulation of alkali metal ions. It observes the presence of symmetry-independent half-molecules of nonactin in the asymmetric unit residing on a crystallographic two-fold axis during crystallization. Also observed is the low-temperature high-precision ordered structure of free nonactin possessing an unfolded and more planar geometry but globular when encapsulating a cation.

Type I polyketide synthases (PKSs) are multifunctional enzymes that are organized into modules, each of which minimally contains a β-ketoacyl synthase, an acyltransferase (AT), and an acyl carrier protein. Here we report that the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 consists of two PKS genes, Inml and InmJ, that encode six PKS modules, none of which contain the cognate AT domain. The only AT activity identified within the Inm gene cluster is a discrete AT protein encoded by InmG. Inactivation of InmG, Inml, or InmJ in vivo abolished LNM biosynthesis. Biochemical characterization of LnmG in vitro showed that it efficiently and specifically loaded malonyl CoA to all six PKS modules. These findings unveiled a previously unknown PKS architecture that is characterized by a discrete, iteratively acting AT protein that loads the extender units in trans to "AT-less" multifunctional type I PKS proteins for polyketide biosynthesis. This PKS structure provides opportunities for PKS engineering as exemplified by overexpressing InmG to improve LNM production. [ABSTRACT FROM AUTHOR]