Biochemical Characterization of a Flavone Synthase I from Daucus carota and its Application for Bioconversion of Flavanones to Flavones.

In this study, we studied the biochemical characterization of flavone synthase I from Daucus carota (DcFNS I) and applied it with flavonoid 6-hydroxylase from Scutellaria baicalensis (SbCYP) to convert flavanones to flavones. The recombinant DcFNS I was expressed in the form of the glutathione-S-transferase fusion protein. Rather than taxifolin, naringenin, pinocembrin, and eriodictyol were accepted as substrates. The optimal temperature and pH for reaction in vitro were 35 °C and 7.5, respectively, and 2-oxoglutarate was essential in the assay system. Co2+, Cu2+, Mn2+, Ni2+, and Zn2+ were not substitutes for Fe2+. EDTA and pyruvic acid inhibited the activity, except for Fe3+. Kinetic analysis revealed that the Vmax and kcat values of the recombinant DcFNS I against naringenin were 0.183 nmol mg-1 s-1 and 0.0121 s-1, and 0.175 nmol mg-1 s-1 and 0.0116 s-1 against pinocembrin. However, the recombinant DcFNS I had a higher affinity for naringenin than pinocembrin, with kM values for each of 0.076 mM and 0.174 mM respectively. Thus, it catalyzed naringenin more efficiently than pinocembrin. Subsequently, using an Escherichia coli and Saccharomyces cerevisiae co-culture system, we successfully converted naringenin and pinocembrin to scutellarein and baicalein respectively. In a synthetic complete medium, the titers of scutellarein and baicalein reached 5.63 mg/L and 0.78 mg/L from 200 mg/L precursors.

Combinatorial Engineering of Upper Pathways and Carotenoid Cleavage Dioxygenase in Escherichia coli for Pseudoionone Production.

Pseudoionone is a valuable intermediate for the flavor industry. However, few attempts have been made to synthesize pseudoionone using biotechnology. In this work, an Escherichia coli strain harboring both the isopentenol utilization pathway (IUP) and mevalonate (MVA) pathway was engineered for the production of the pseudoionone precursor lycopene, which increased the titer of lycopene to approximately 20-fold the yield obtained by the original MEP pathway. Subsequently, the crucial limiting step of carotenoid cleavage dioxygenases (CCDs) was evaluated and optimized. The most effective upstream module for pseudoionone synthesis in E. coli was determined to be MnCCD1 from Morus notabilis without a GST-tag and under the control of the tac promoter. Finally, the highest pseudoionone production achieved 20.61 mg/L under the optimum fermentation conditions in a shake flask. This study provided an efficient approach for pseudoionone production and advanced our understanding of the characteristics of the CCD family for the biosynthesis of aroma compounds.

Confirmation of Gelsemium elegans poisoning by UHPLC-MS/MS analysis of koumine, gelsemine, and gelsenicine in hair.

A sensitive, accurate, simple, and rapid analytical UHPLC-MS/MS method was developed for identification and quantification of koumine, gelsemine, and gelsenicine in human hair. Approximately 10 mg of hair was extracted with methanol by cryogenic grinding. The limits of detection (LODs) ranged from 1 to 5 pg/mg, and the limits of quantitation (LOQs) ranged from 2 to 10 pg/mg. The method was linear over a concentration range from the LOQs to 1000 pg/mg, and the linear correlation (R2) of the calibration curves was above 0.998 for all three analytes. The bias varied from -6.5-13.1%, while the intra- and inter-day precision relative standard deviation (RSD) values were 4.3-12.4% and 3.7-13.2%, respectively. Recoveries ranged from 79.3% to 103.5%, and matrix effects ranged from 74.3% to 105.5%. The described method was used for the quantitative determination of koumine, gelsemine, and gelsenicine in a human hair sample from a Gelsemium elegans poisoning case. The highest concentrations of koumine, gelsemine, and gelsenicine were 27.2, 18.1, and 4.2 pg/mg, respectively, and corresponded to the segment associated with the ingestion period. To our knowledge, this is the first study to describe hair analysis in a G. elegans poisoning case and to provide quantitative toxicological findings.

Overexpression and Characterization of a Novel Plant Carotenoid Cleavage Dioxygenase 1 from Morus notabilis.

Synthesis of ß-ionone in microbial cell factories is limited by the efficiency of carotenoid cleavage dioxygenases (CCDs). To obtain genes responsible for specific cleavage of carotenoids generating ß-ionone, a novel carotenoid cleavage dioxygenase 1 from Morus notabilis was cloned and overexpressed in Escherichia coli. The MnCCD1 protein was able to cleave a variety of carotenoids at the positions 9, 10 (9', 10') to produce ß-ionone, 3-hydroxy-4-oxo-ß-ionone, 3-hydroxy-ß-ionone, and 3-hydroxy-α-ionone in vitro. MnCCD1 could also cleave lycopene and ß-carotene at the 9, 10 (9', 10') bind bond to produce pseudoionone and ß-ionone, respectively, in E. coli accumulating carotenoids. The enzyme activity of MnCCD1 was reached 2.98 U/mL at optimized conditions (temperature 28 °C, IPTG 0.1 mM, induction time 24 h). The biochemical characterization of MnCCD1 revealed the optimal activities were at pH 8.4 and 35 °C. The addition of 10 % ethanol could increase enzyme activity at above 15 %. However, an obvious decline was observed on enzyme activity as the concentration of Fe2+ increased (0-1 mM). The Vmax for ß-apo-8'-carotenal was 72.5 U/mg, while the Km was 0.83 mM. The results provide a foundation for developing the application of carotenoid cleavage dioxygenases as biocatalysis and synthetic biology platforms to produce volatile aroma components from carotenoids.

Field-amplified sample injection in capillary zone electrophoresis for the pharmacokinetic research of trace risperidone and its major metabolite 9-hydroxyrisperidone in beagle dogs.

This article describes a method for the simultaneous quantitation of risperidone and its major metabolite, 9-hydroxyrisperidone, in beagle dog plasma by field-amplified sample injection in capillary zone electrophoresis. The separation was carried out at 25°C in a 48 cm × 75 µm fused-silica capillary with an applied voltage of 20 kV using 60 mM NaH2 PO4 buffer (pH 3.6). The detection wavelength was 280 nm. Clean-up and preconcentration of plasma samples were conducted by 96-well formatted liquid-liquid extraction. In this study, this stacking technique provided a sensitivity enhancement of approximately 158 to 188 fold compared with the same sample without stacking. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, and extraction recovery. Calibration curves exhibited good linearity (r2  > 0.995) over a wide concentration range of 2.5 to 200 ng/mL for both risperidone and 9-hydroxyrisperidone. The intra- and interday precisions at the three quality control levels were less than 11.40%. The intra- and interday accuracies ranged from 87.90 to 107.17% for risperidone and from 88.43 to 105.92% for 9-hydroxyrisperidone. All validation data were within the required limits. In conclusion, the method developed was successfully applied to pharmacokinetic studies of risperidone and 9-hydroxyrisperidone in beagle dogs.

Validation of a universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry methodology for the quantification of pyrazinamide, ethambutol, protionamide, and clofazimine in different biological matrices.

A novel and potent anti-tuberculosis drug combination pyrazinamide (PZA), ethambutol (EMB), protionamide (PTO), and clofazimine (CFZ) that rapidly kills Mycobacterium tuberculosis (Mtb) in the lungs has been identified using the artificial-intelligence-enabled parabolic response surface approach. A universal and highly sensitive two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) method for the simultaneous determination of PZA, EMB, PTO, and CFZ in various biological samples in different states (liquid samples: plasma, bile, and urine; solid samples: tissue and feces) using simple pretreatment was established and validated. For the first dimension of this column-switching arrangement, the automated purification and enrichment of the drugs were achieved on a Polar-RP column. The subsequent analytical separation was performed on an Agilent Zorbax SB-Aq column, and the total loop time was 7.5 min. The positive-ionization mode with multiple reaction monitoring was used for detection. The sensitivity was good with no carry-over detected, and the lower limit of quantification ranged from 100 to 500 pg/mL. This quantification method was fully validated and proved to be robust in accordance with US Food and Drug Administration guidelines. High recoveries (85.3-111.4%) and accuracies (92.1-109.3%), together with high precision values (0.5-13.8%), were verified in all matrices. All standard curves showed favorable linearities with r2 > 0.995. This validated method was applied to study plasma pharmacokinetics, tissue distribution, and excretion in Sprague-Dawley rats after oral administration of the drug combination.