Construction of Inducible Genetic Switch for the Global Regulator WblA To Sustain Both Overproduction of Tiancimycins and On-Demand Sporulation in Streptomyces sp. CB03234.

The complex life cycle of streptomycetes is closely related to their secondary metabolisms, all controlled by cascade regulations. Tiancimycins (TNMs) are ten-membered enediynes possessing great potential for antitumor drug development. However, their low yields in Streptomyces sp. CB03234 have greatly limited subsequent studies. Through transcriptome analysis and genetic characterization, we proved that WblA is one pivotal global regulator to repress the biosynthesis of TNMs. The deletion of wblA could significantly enhance the production of TNMs, but also abolish the sporulation in CB03234. By constructing the NitR-ε-caprolactam inducible genetic switch, the expression of wblA was governed in CB03234-NRW, thereby sustaining the overproduction of TNMs and recovering the normal sporulation upon induction, which were practical for the scaled-up production of TNMs. Considering the prevalence and conserved regulatory roles of WblA in streptomycetes, our developed strategy shall provide an effective and practical approach to facilitate titer improvement and discovery of natural products.

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D.

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.

Identification of the active components in Shenmai injection that differentially affect Cyp3a4-mediated 1'-hydroxylation and 4-hydroxylation of midazolam.

Shenmai injection (SMI) is a popular herbal preparation that is widely used for the treatment of atherosclerotic coronary heart disease and viral myocarditis. In our previous study, SMI was shown to differentially affect CYP3A4-mediated 1'-hydroxylation and 4-hydroxylation of midazolam (MDZ). The present study was conducted to identify the active components in SMI responsible for the differential effects on MDZ metabolism, using in vitro incubation systems (rat and human liver microsomes and a recombinant CYP3A4 system) to measure 1'-hydroxylation and 4-hydroxylation of MDZ. First, different fractions of SMI were obtained by gradient elution on an solid phase extraction system and individually tested for their effects on MDZ metabolism. The results demonstrated that lipid-soluble constituents were likely to be the predominant active components of SMI. Second, the possible active components were gradually separated on an high-performance liquid chromatography system under different conditions and individually tested in vitro for their effects on MDZ metabolism. Third, the active component obtained in the above experiment was collected and subjected to structural analysis, and identified as panaxytriol (PXT). Finally, it was validated that PXT had significant differential effects on 1'-hydroxylation and 4-hydroxylation of MDZ in various in vitro systems that were similar to those of SMI. We conclude that PXT is the constituent of SMI responsible for the differential effects on CYP3A4-mediated 1'-hydroxylation and 4-hydroxylation of MDZ.

Structural and conformational features relevant to the anti-tumor activity of calicheamicin γ 1I.

The structural and conformational features of the potent 10-membered enediyne-containing calicheamicin γ 1I that account for its remarkable DNA site-specific binding and cleavage are reviewed. A variety of spectroscopic and biophysical techniques were used to gain insight into the binding and stereospecific DNA cleavage of this potent antitumor agent. These include gel-shift cleavage assays, atom transfer NMR experiments, drug-DNA conformational studies, circular dichroism, and capillary electrophoresis. Computational descriptions are described for the DNA binding and cleavage of calicheamicin and its activated transient intermediates based on density functional and molecular mechanics calculations. In addition, the structure and clinical utility of calicheamicin immunoconjugates for antibody-targeted chemotherapy is presented.

DFT/NMR integrated approach: a valid support to the total synthesis of chiral molecules.

The analysis of the configuration of kedarcidin chromophore and palau'amine through quantum chemical calculation of Js and chemical shifts suggests a fast and convenient quantum chemical approach that can be applied prior to proceeding to the total synthesis of complex natural compounds in order to avoid loss of time and resources employed in the total synthesis of wrong diastereoisomers.