[Metabolic engineering study on biosynthesis of 4-hydroxybenzyl alcohol from L-tyrosine in Escherichia coli].

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.

[Gene cloning and prokaryotic expression of glycosyltransferase from Ligustrum quihoui].

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%), ß sheet(12.1%) and random coil(49.9%), and tertiary structure was constructed by peptide chain folding to form two face-to-face α/ß/α 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 in vitro and clarifying the functional mechanism of enzyme.