| 英文摘要 |
Natural medical plants have historically proven their importance in human medicine history, and now still represent an important pool for identifying and isolating bioactive natural products for novel drug discovery. Glycosides, one of the major groups of natural products, have gained much interest for their high solubility and various bioactivities, such as salidroside and ginsenosides. But the rarity of many valuable natural plants and the complexity of various glycosides in natural plants make it difficult to isolate enough glycoside monomer. With the last development of bioinformatics and biotechnology, enzymatic transformation and synthetic biology have shown great advantage in the biosynthesis of many important natural products. Glycosylation is the final step of the biosynthesis of many glycosides, which is mainly catalized by various glycosyltransferases. Thus, it is very important to screen glycosyltransferases for the biosynthesis of natural glycosides. Crocins are the major active ingredients of natural medical plant Crocus sativus, which is famous for its anti-tumor, antioxidation, and cardiovascular protection properties. But the biosynthesis of crocins was restricted by the lack of efficient crocetin glycosyltransferases. In this study, to provide efficient crocetin glycosyltransferase for crocins synthesis, we mined a microbe-derived crocetin glycosyltransferase (Bs-GT) based on the combined analysis of N-terminal and PSPG motif of plant-derived crocetin glycosyltransferases. Through optimization of mRNA secondary structure and inducing conditions, Bs-GT was efficiently expressed in engineered E.coli. Bs-GT protein was purified by Ni-NTA affinity chromatography and its enzymatic property was characterized. The glycosylation activity of Bs-GT was further improved by protein engineering. Non-aqueous two-enzyme coupled reaction system was established to improve the solubility of substrate crocetin and reduce the inhibition effect of high concentration of UDP, and efficient enzymatic synthesis of crocins was achieved in this reaction system. (1)Mining of crocetin glycosyltransferase across plant-microbial kingdom: The key motifs involved in the binding of crocetin to plant-derived crocetin glycosyltransferases UGT75L6 and Cs-GT2 were predicted by multiple sequence alignment, homologous modeling and in slico docking analysis. The combined analysis of N-terminal and PSPG motif was applied to screen crocetin glycosyltransferases, and seven candidates (Bs-GT, Bc-GT A, Bc-GTB, Bl-GT1, Bl-GT5, Bp-GT1, Fg-GT) were mined. The seven glycosyltransferases were cloned and expressed in E.coli-BL21(DE3). Whole-cell transformation system was established and two glycosyltransferases (Bs-GT and Bc-GTA) were confirmed to be capable of glycosylating crocetin to crocins. Compared to 5c-GTA, Bs-GT showed higher glycosylation activity towards crocetin, with a conversion rate of 35%. Bs-GT and 5c-GTA were the first microbial glycosyltransferases that could glycosylate crocetin to crocins. (2)Efficient expression and purification of 5s-GT: The 5’ mRNA secondary structure of various Bs-GT recombinant plasmids was predicted by RNA Structure online service. Since the recombinant plasmid pET-28a-5s-GT-Nhe I had higher △G (-3.2kcal/mol) and no complex stem-loop structure, plus the initiation codon ATG was located in the single chain, pET-28a-5s-GT-Nhe I was chosed for efficient expression of Bs-GT in E.coli. The recombinant E.coli- pET-28a-5s-GT-Nhe I was constructed and the inducing conditions were optimized. The best inducer was 25μmol/L IPTG, and the best inducer adding time was when OD₆₀₀ reached 0.8. After induced at 20 ℃ for 24h, the maximum glycosylation activity of Bs-GT reached 197U/L. Bs-GT was then purified by Ni-NTA affinity chromatography and the glycosylation activity of Bs-GT towards crocetin was determined to be 2.26U/㎎. (3)Enzymatic characterization and substrate specificity determination of 5s-GT: The optimized temperature of 5s-GT was determined as 30℃, and 5s-GT remained stable under 25 ℃. The optimized pH was determined as 9.5, and remained high activity at alkali conditions (pH8.0-10.0). 5s-GT is a typical GT-B fold glycosyltransferase and its glycosylation activity is independent on metal ions. Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺ activated 5s-GT slightly, and Ag⁺, Zn²⁺ would inhibit the glycosylation activity of Bs-GT.Bs-GT showed good tolerance to organic solvents, and remained more than 90% initial activity when incubated with 10% (v/v) of DMSO or DMF for 4h. The substrate specificity determination indicated that Bs-GT also showed high glycosylation activity of some flavonoids like quercetin, kaempferol, and resveratrol. When catalyzed by Bs-GT, with UDPG as sugar donor, about 80% of crocetin (0.02mmol/L) could be glycosylated to crocin-5 and crocin-3 after 2h. (4)Protein engineering of Bs-GT. Homology model of Bs-GT was constructed and its active pocket was predicted. Through in slico docking analysis and alanine scanning, Hisl7, Asp328, and Gln329 were determined to be crucial for glycosylation activity, and Thr11A and Phe242A were determined to be beneficial for crocetin glycosylation Saturated mutation at Thr11 was then carried out. Results indicated that T11G mutation showed the highest glycosylation activity of Bs-GT towards crocetin Then further saturated mutation at Phe242 based on T11G was caonducted, and the most effective mutant T11G-F242Y was obtained. The enzymatic proterity of T11G-F242Y was characterized. Compared to wild-type 5s-GT, T11G mutation had little effects on the temperature and pH preference. But the K〓 towards crocetin was decresed. In slico T11G mutation simulation and docking analysis showed a decrease in binding energy of crocetin onto T11G-F242Y than wild Bs-GT. Compared to wild Bs-GT, T11G-F242Y mutant showed a 1.41-fold higher glycosylation activity towards crocetin, and the total glycosylation rate was increased from 81 % to 89%. (5)The construction of non-aqueous two-enzyme coupled glycosylation system: The non-aqueous system was established through adding DMSO into the reaction buffer system. Bs-GT showed high activity and stability in 10% DMSO-buffer system Adding DMSO into the reation system not only increased the solubility of crocetin dramatically, but also increased the substrates selectivity of Bs-GT, which would benefit to the regulation of crocetin glycosylation products. By adding sucrose synthetase(At-ss from Arabidopsis thaliana) into the glycosylation system, the Bs-GT and At-SS coupled reaction system for crocetin glycosylation was established. The reaction condition was further optimized. The reaction system showed highest glycosylation activity under pH8.0 Gly-NaOH buffer, with 1mmol/L UDP and 300mmol/L sucrose. The optimized adding ratio of 5s-GT and At-SS was determined as 1.5:1. The construction of Bs-GT and At-SS coupled reaction system not only dramatically reduced the inhibition effect of high concentration of UDP, but also reduced the cost significantly as only sliht amount of UDP is needed. The non-aqueous catalysis and glycosyltransferase-sucrose synthetase coupled catalysis system great potential for biosynthesis of crocins due to its higher selectivity and tower cost. And through fed-batch reaction, a maxmium yield of 4.88mmol/L crocin-3 and crocin-5 was achieved. Key words: Crocetin; Crocins; Glycosyltransferase; N-terminal motif; PSPG motif; Mining across kingdoms; efficient expression; protein engineering; Non-aqueous two--enzyme coupled system.
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