ENGINEERING BACILLUS SUBTILIS FOR PRODUCTION ANTIMALARIA ARTEMISININ AND ANTICANCER PACLITAXEL PRECURSORS

• Main Author: Pramastya, Hegar
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/48628
• Institution: Institut Teknologi Bandung

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. With its generally recognized as safe status and its high capacity of endogenous pathway, Bacillus subtilis holds important position as a candidate for terpenoid cell factory. However, higher scale production of natural product requires stable expression of a biosynthetic pathway enzymes. The aim of this study was to construct B. subtilis strains capable of producing high titer of amorphadiene and taxadiene by upregulating endogenous methyl erythritol phosphate (MEP) pathway or installing the heterologous mevalonate (MVA) pathway. First, the stable expression of extra endogenous pathway was established by construction of a regulated synthetic operon consisting eight genes of MEP pathway together with ispA gene encoding farnesyl diphosphate synthase. The operon constructed in theta replicating plasmid was segregationally and structurally more stable compared to rolling circle plasmid which eventually brought high production of C30 carotenoid in B. subtilis carrying crtM and crtN genes up to 20 mg/gr dried cell weight. The stable synthetic operon was then utilized for production taxadiene as a dedicated precursor of paclitaxel. The diterpene taxadiene requires geranylgeranyl diphosphate which supplied by the geranylgeranyl diphosphate synthase (encoded by crtE) of Pantoea ananatis. In addition to generating appreaciable amount of taxadiene (up to 17.8 mg/L), the research also showed the stable co-expression between two theta replicating plasmids that beneficial for further research in metabolic engineering of B. subtilis. Production of high amount of amorphadiene is required to ensure economically feasible and sustainable production of artemisinin. Amorphadiene synthase (Ads) was poorly expressed in B. subtilis. N-terminal modification of Ads by fusion with optimized green fluorescent protein (Gfp) facilitated higher expression of the protein. The combination of MEP pathway overexpression, Ads improved expression and medium optimization brought amorphadiene production up to 416 ± 15 mg/L in flask scale. Precursor supplementation and pH buffering through pyruvate and potassium phosphate supplementation respectively highlighted the significant of main carbon flux and medium pH control. For the next level, mevalonate (MVA) pathway was constructed to improve the amorphadiene production. The construction combined the upper part of Enterococcus faecalis and lower part of Streptococcus pneumoniae mevalonate pathway. Expression of the upper part of the pathway enabled the bacterium to produce mevalonate. However the whole pathway was not self-sufficient that still required mevalonate supplementation for high production of amorphadiene. MVA pathway heterologous pathway expression together with mevalonate supplementation and upregulation of isopentenyl diphosphate isomerase (Idi) and farnesyl diphosphate synthase (IspA) up to 800 mg/L. Thus, it implied the functionality of lower part of MVA pathway. Meanwhile upper MVA pathway still has to be improved. Taken together, it has been a milestone that B. subtilis can be configured as prospective terpenoid cell factory and might become alternative solution in production of natural product in sustainable manner.