Engineering Bacillus subtilis for Production of Antimalaria Artemisinin and Anticancer Paclitaxel Precursors

Terpenoids are natural products with diverse applicability in foods, cosmetics, and pharmaceuticals industries. Among them are antimalaria artemisinin and anticancer paclitaxel. The research in the thesis is aimed to engineer Bacillus subtilis, a gram-positive bacterium, for high production of amorphadiene and taxadiene, the precursor of antimalaria artemisinin and anticancer paclitaxel respectively. With its generally recognized status as safe bacterium, B. subtilis can be a more economic host for the production. The bacterium itself own MEP pathway as the endogenous pathway to produce precursors of terpenoids but insufficient yet to generate high titter of terpenoids. Here the author improved the terpenoid production by upregulating the endogenous pathway in a stable manner. A stable synthetic cluster of genes of the pathway was employed to produced high titer of three different terpenoids including taxadiene and amorphadiene. Indeed, the genetic manipulation included the heterologous expression of terpene synthase as the downstream enzyme in the terpenoids biosynthetic pathways. Other measures including protein fusion leading higher expression of amorphadiene synthase and medium optimization were employed. At the end, as a proof of concept, an alternative heterologous MVA pathway was built and expressed, providing more precursors supply for higher amorphadiene production. The multiapproach shown in thesis would be applicable to produce considerable yield of terpenoids, not only amorphadiene and taxadiene.