| Glycyrrhetinic acid(GA),a kind of oleanolic triterpenoid,has extensive pharmacological activities such as liver protection,anti-virus,anti-cancer and so on.At present,GA is mainly obtained either by extraction from licorice plants directly or chemical synthesis from the hydrolysis of glycyrrhizic acid coming from licorice plants.Both of the above methods consume a lot of acid,alkali and freshwater,easily lead to environmental pollution and ecological damage and other serious problems.Also,the licorice growth cycle is long and consume a lot of arable land,coupled with licorice excavation strictly controlled by the government policies which greatly limit the wide applications of GA in chemical,pharmaceutical and other areas.In order to improve the titer of GA,we intorduced the whole GA synthetic pathway of licoice plants into yeast using synthetic biology and metabolic engineering strategies combined with transcriptomics analysis.High efficient biosynthesis of GA was finally achieved via pathway optimization and transcriptional regulation in engineered Saccharomyces cerevisiae.The main results obtained in this studty are as follows:(1)Genes related to the biosynthesis of GA were introduced into yeast,including ?-amyrin synthase gene,CYP450 gene and CYP450 reductase gene.The successful co-expression of these genes on mutiple plasmids contributed to the synthesis of GA with a titer of 20 ?g/L in engineered yeast.A variety of GA precursors such as 11-hydroxy-?-amyrin,11-oxo-?-amyrin,and 30-hydroxy-11-oxo-?-amyrin as well as some byproducts such as 30-hydroxy-?-amyrin and 11,30-hydroxy-?-amyrin were identified by GC-MS analysis.(2)The transcriptions database of Glycyrrhiza and genome databases of related plants were studied to serach for new enzymes with similar functions of CYP450 s,which is more efficient and specific compared with the original one.A number of enzymes were obtained employing methods including Nt-Identity,amino acid similarity,functional annotation and gene clusters mining,which can catalyze ?-amyrin or 11-oxo-?-amyrin into products including ?-boswellic acid,24-hydroxy-?-amyrin and the like.At the same time,we also identified a number of enzymes that can catalyze 11-oxo-?-amyrin,such as CYP72A63,Unigene33312,Unigene27475 and so on.(3)The synthetic pathway of GA was optimized in yeast.The production of GA increased 1.25-fold when the synthetic pathway were integrated into yeast genome.INVSc1 strain was proved as the best host for GA production,increasing 1.32-folds of its titer.The replacement of key enzyme enhanced the production of GA for about 1.26-folds compared to that of the control strain.The titers of ?-amyrin and ergosterol were increased by 2.25 and 1.37-folds,respectively,by overexpressiong tHMG1 and upc2-1.By increasing copy numbers of Uni25647(Unigene25647),the production of 11-oxo-?-amyrin(precursor of GA)was improved to 108 mg/L.The production of GA was further improved to a yield of 517 ?g/L by CPRs screening from different plants.Meanwhile,we found that the ratio of CYP450 vs CPR had a great effect on the catalytic activity of CYP450 s.Finally,Uni25647,CYP72A63 and GuCPR1 were selected as the optimal combination to improve the production of GA after the screening of various gene combinations,and the finally obtained titers was 7.4 mg/L.(4)Fermentation optimization was carried out to enhance GA production.The production of 11-oxo-?-amyrin was increased to 187 mg/L in the glucose fed-batch fermentation,which is 2494-fold higher than the titer that ever reported.In the ethanol fed-batch process,the maximum production of 11-oxo-?-amyrin was 80 mg/L,resulting in an improvement of GA to a titer of 18.9 mg/L,which is 947-fold higher than the titer that ever reported.Compared with glucose-feeding,ethanol-feeding can significantly improve the production of GA. |
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