Metabolic Engineering of Bacillus subtilis Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production

Abstract

Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) <i>Bacillus subtilis</i> 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering <i>B. subtilis</i> as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the <i>B. subtilis</i> genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (<i>dxs</i>, <i>ispD</i>, <i>ispF</i>, <i>ispH</i>, <i>ispC</i>, <i>ispE</i>, <i>ispG</i>) together with <i>ispA</i> (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the <i>crtE</i> gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of <i>Pantoea ananatis</i>) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of <i>B. subtilis</i>. The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in <i>B. subtilis</i>. We determined that the expression of GGPPS through the <i>crtE</i> gene is essential for the formation of sufficient precursor, GGPP, in <i>B. subtilis</i> as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.