Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli / 中国中药杂志

As an important active ingredient in the rare Chinese herb Gastrodiae Rhizoma and also the main precursor for gastrodin biosynthesis, 4-hydroxybenzyl alcohol has multiple pharmacological activities such as anti-inflammation, anti-tumor, and anti-cerebral ischemia. The pharmaceutical products with 4-hydroxybenzyl alcohol as the main component have been increasingly favored. At present, 4-hydroxybenzyl alcohol is mainly obtained by natural extraction and chemical synthesis, both of which, however, exhibit some shortcomings that limit the long-term application of 4-hydroxybenzyl alcohol. The wild and cultivated Gastrodia elata resources are limited. The chemical synthesis requires many steps, long time, and harsh reaction conditions. Besides, the resulting by-products are massive and three reaction wastes are difficult to treat. Therefore, how to artificially prepare 4-hydroxybenzyl alcohol with high yield and purity has become an urgent problem facing the medical researchers. Guided by the theory of microbial metabolic engineering, this study employed the genetic engineering technologies to introduce three genes ThiH, pchF and pchC into Escherichia coli for synthesizing 4-hydroxybenzyl alcohol with L-tyrosine. And the fermentation conditions of engineering strain for producing 4-hydroxybenzyl alcohol in shake flask were also discussed. The experimental results showed that under the conditions of 0.5 mmol·L~(-1) IPTG, 15 ℃ induction temperature, and 40 ℃ transformation temperature, M9 Y medium containing 200 mg·L~(-1) L-tyrosine could be transformed into(69±5)mg·L~(-1) 4-hydroxybenzyl alcohol, which has laid a foundation for producing 4-hydroxybenzyl alcohol economically and efficiently by further expanding the fermentation scale in the future.

Research progress on biosynthesis of gastrodin / 中草药

Gastrodin is a major active ingredient in Gastrodia elata, which is one of the traditional rare medicinal herbs in China. It has lots of pharmacological activities, such as reducing blood pressure, anti-epileptic, anti-tumor, and protecting nerve, etc. With the increasing demand for gastrodin in the market and the inherent problems of traditional methods on obtaining gastrodin, new methods are urgently needed to solve various difficulties in the actual production of gastrodin. Biosynthesis is a new method instead of the traditional acquisition method, and has made great progress and achievements in gastrodin acquisition. Therefore, it is necessary to systematically review the biosynthesis of gastrodin from three aspects of biosynthetic pathway, plant transformation and microbial transformation in this paper, hoping to provide valuable reference for further improvement and perfection of gastrodin production method in the future to meet public increasing demand for gastrodin.

Gene cloning and prokaryotic expression of glycosyltransferase from Ligustrum quihoui / 中国中药杂志

According to the previous results from transcriptome analysis of Ligustrum quihoui, a glycosyltransferase gene(xynzUGT) was cloned by rapid amplification of cDNA ends(RACE). The full length cDNA of xynzUGT was 1 598 bp, consisting of 66 bp 5'-UTR, 1 440 bp ORF and 92 bp 3'-UTR. The ORF encoded a 480 amino-acid protein(xynzUGT) with a molecular weight of 54 826.67 Da and isoelectric point of 5.82. The structure of enzyme was analyzed by using bioinformatics method, the results showed that the primary structure contained a highly conserved PSPG box of glycosyltransferase, the secondary structure included α helix(38%), sheet(12.1%) and random coil(49.9%), and tertiary structure was constructed by peptide chain folding to form two face-to-face domains(often referred to as a Rossmann domains), between which a substrate binding pocket is sandwiched. The phylogenetic tree analysis indicated that xynzUGT might catalyze glycosylation of phenylpropanoids, such as tyrosol. Further simulation experiment of molecular docking between enzyme and tyrosol showed that Gly138 and Ser285 located in the binding pocket interacted with tyrosol by hydrogen bonding. SDS-PAGE analysis exhibited that the prokaryotic expression system successfully expressed recombinant xynzUGT with molecular weight of 58 370.57 Da, but it exists in the form of non-soluble inclusion bodies. Using the molecular chaperone and enzyme co-expression method, the soluble expression was promoted to some extent. The above works laid the foundation for further studying on enzymatic reaction and clarifying the functional mechanism of enzyme.

Inhibition of Jingzhaotoxin-V on Kv4.3 channel / 生理学报

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.