Construction Of Engineered Yeast Strains For Biosynthesis Of Artemisinin Precursors And Biotransformation Of Fermentative Products

The chemical nature of artemisinin is the sesquiterpene lactone peroxide, which is an antimalarial monomer isolated and purified from the traditional Chinese medicinal herb Artemisia annua by Chinese scientists in 1970's. As a kind of secondary metabolites, artemisinin scarcely accumulates in A. annua. Additionally, the natural resource of A. annua is extremely rare; plantation of A. annua occupys a large area of cultivated fields; and chemical biosynthesis of artemisinin cannot be readily industrialized due to involving a complicated, costly and toxic process. In consequence, production of artemisinin in engineered microorganisms has been attempted worldwide although no artemisinin per se rather than artemisinin precursors has been synthesized in any engineered microorganisms up to the present. With success in construction of the engineered yeast that expresses amorphadiene synthase gene (ADS) of A. annua and production of the artemisinin precursor amorphadiene, we further introduce two other necessary genes for artemisinin biosynthesis, cytochrome P450 monooxygenase gene (CYP71AV1) and artemisinic aldehyde double-bond reductase gene (DBR2), into the yeast with ADS. The presence of the recombinant plasmid containing A. annua genes in the engineered yeast was verified by PCR, double digestion and re-sequencing, while the functional expression of recombinant artemisinin biosynthetic genes was validated by phenotyping and RT-PCR. GC-MS analysis of the acidic water phase indicated that fermtative products in three-type of engineered yeast strains were more than those in the wild-type yeast, in which products in the three-gene-transferred yeast were more than those in the two-gene-transferred yeast, whereas products in the two-gene-transferred yeast were more than those in the one-gene-transferred yeast, demonstrating that the expression of different artemisinin biosynthetic genes in engineered yeast cells led to the biosynthesis of distinct artemisinin precursors. GC-MS analysis of the organic phase confirmed the presence of amorphadiene with 1.7μg/mL. To enzymatically convert the yeast-produced artemisinin precursors into artemisinin, biotransformation by incubation of the hexane extract from each engineered yeast with the cell-free enzyme mixture of cold-acclimed A. annua was carried out and artemisinin content was monitored by HPLC. As results, artemisinin content in biotransformation products of transgenic yeast strains was 3.28mg/mL, accounting for approximately 10 folds higher than the control, which may represent one of the most enhanced artemisinin production projects of metabolic engineering aiming at artemisinin overproduction. The creative outcomes of the present study are that:(1) a novel engineered yeast that simultaneously express three artemisinin biosynthetic genes has been available; (2) artemisinin precursors produced by engineered yeast strains have been converted to artemisinin by the biotransformation procedure; (3) a "two-step method" for production of artemisinin by the interplay between the microorganism (yeast) and the plant(A. annua) has been established. The prospective applications of these achievements are providing thereotic supports for harnessing dual merits of the scaled industrial fermentation and the economic agricultural plantation to realize enhanced artemisinin production and to resolving the predicment in the insufficient artemisinin supply.