Biotransformation Of Glycosylated Derivatives Of Curcumin And Its Analogues

Curcuma longa is a commonly used traditional Chinese medicine,which has been published in the past editions of Pharmacopoeia of the People’s Republic of China and has a wide range of pharmacological effects and good safety.It has a wide history of application in China and in Asia.Curcumin is the main component of turmeric.Modern pharmacological studies show that curcumin has anti-inflammatory,anti-oxidation,anti-tumor,hepatoprotective,immune regulatory,and other pharmacological effects.However,the physical property of low polarity and insoluble in water are important limiting factors of curcumin in medicine and clinical application.Because of low bioavailability and poor absorption,the forms of medicinal substances in the body of curcumin have been debated for a long time.The metabolites of curcumin in the human body are mainly glucuronidated curcumin,a phase Ⅱ metabolite.Therefore,exploring the pharmacological activity of glucuronidated curcumin is an important part of confirming the forms of medicinal substances in the body.As the biogenic synthetic pathways of glycoside compounds widely existing in nature,glucuronidation and glucosylation are effective methods to increase the solubility of parent compounds.Therefore,the construction of a biotransformation system to conduct glucuronidation and glucosylation biotransformation of curcumin is an effective means to study the forms of medicinal substances in the body of curcumin,improve its physical and chemical properties,and increase its druggability.In this experiment,we used methods of synthetic biology to construct suitable vectors,constructed exogenous protein expression systems in commonly used engineering bacteria such as Escherichia coli and Saccharomyces cerevisiae,and constructed glucuronidation and glucosylation biotransformation systems,using whole-cell catalysis,cell-free catalysis,and other methods to study the glucuronidation and glucosylation biotransformation of curcumin and a variety of polyphenols.The specific research content and results are as follows:1.Construction of glucuronidation system in E.coliIn this chapter,we selected different vectors to construct the UDP glucuronosyltransferase and UDP-glucose 6-dehydrogenase expression system.We used molecular chaperones,maltose-binding protein,and other auxiliary means to help the lipophilic human UDP glucuronosyltransferase UGT1A8 expression and folding,and also optimized protein expression conditions to increase the expression level of the two proteins in E.coli.CRISPRCas9 was used to seamlessly knock out the UDP-glucuronic acid dehydrogenase gene arn A on the chromosome of E.coli to optimize the metabolic flow of the biotransformation substrate UDP glucuronic acid and increase the sugar supply.A variety of polyphenolic substrates were used to explore the substrate spectrum of UGT1A8.We also tried a variety of biotransformation conditions and used whole-cell and cell-free systems to catalyze the substrate for glucuronidation to eliminate the influence of solvents,temperature,metal ions,substrate transport,and other conditions on the substrate biotransformation.In the end,no glucuronidate product was detected,which may be because of the UGT1A8 protein cannot be folded correctly in prokaryotes that lack the necessary organelles.Although the experiment in this chapter did not succeed in obtaining glucuronidation biotransformation products,it was a summary of experience for the subsequent expression of eukaryotic-derived proteins in prokaryotes.2.Construction of glucuronidation system in S.cerevisiaeUDP glucuronosyltransferase and UDP-glucose 6-dehydrogenase expression systems were constructed in S.cerevisiae to try to solve the problem that eukaryotic-derived proteins cannot be folded correctly in prokaryotes.By using self-replicating plasmids to induce the expression of the target protein,try to perform glucuronidation biotransformation experiments on various substrates such as curcumin,tetrahydrocurcumin,alizarin,umbelliferone,resveratrol,naringenin,emodin,and rhein.Finally,the production of glucuronidated alizarin was detected,indicating that the yeast glucuronidated biotransformation system was successfully constructed.However,the glucuronidated curcumin was not detected in this biotransformation system.3.Biotransformation of glucosylated curcumin and its analogsA glycosylation biotransformation system was constructed in E.coli,and glucosyltransferase from Catharanthus roseus was heterologously expressed to perform glycosylation biotransformation on curcumin and its reduction products.In this experiment,the reducing activity of S.cerevisiae on tetrahydrocurcumin was discovered for the first time,and the hexahydrocurcumin was produced in large quantities by S.cerevisiae,which laid the material foundation for the biotransformation of glucosylation.In this experiment,glucosylated tetrahydrocurcumin and glucosylated hexahydrocurcumin were isolated and identified for the first time.Glucosylated curcumin was successfully prepared.It was found that this biotransformation system has glucosylation activity on dihydrocurcumin and octahydrocurcumin,too.The experiments in this chapter opened up new research ideas for solving the problem of curcumin’s physical and chemical properties,laid a material foundation for studying the pharmacological activities of glucosylated curcuminoids,and provided a transformation system and research experience for the preparation of rare glycoside natural products.Conclusion:1.Heterologous expressed of human UDP glucuronosyltransferase UGT1A8 and murine UDPglucose 6-dehydrogenase Ugd in S.cerevisiae.The production of glucuronidated alizarin was detected,and the biotransformation system of glucuronidation was successfully constructed in S.cerevisiae.2.The glucosyltransferase from C.roseus was heterologously expressed in E.coli,and glucosylated tetrahydrocurcumin and glucosylated hexahydrocurcumin were prepared and identified for the first time through whole-cell biotransformation.