Dataset Information

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) Bacillus subtilis 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering B. subtilis 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 B. subtilis genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (dxs, ispD, ispF, ispH, ispC, ispE, ispG) together with ispA (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the crtE gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of Pantoea ananatis) 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 B. subtilis. 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 B. subtilis. We determined that the expression of GGPPS through the crtE gene is essential for the formation of sufficient precursor, GGPP, in B. subtilis 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.

SUBMITTER: Abdallah II

PROVIDER: S-EPMC6391936 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Abdallah Ingy I II Pramastya Hegar H van Merkerk Ronald R Sukrasno Quax Wim J WJ

Frontiers in microbiology 20190220

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) Bacillus subtilis 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering B. subtilis as a cell factory for the production of ...[more]

PMID: 30842758

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Project description:The cell division protein GpsB is a regulator of the penicillin binding protein A1 (PBP A1) in the Gram-positive human pathogen Listeria monocytogenes Penicillin binding proteins mediate the last two steps of peptidoglycan biosynthesis as they polymerize and cross-link peptidoglycan strands, the main components of the bacterial cell wall. It is not known what other processes are controlled by GpsB. L. monocytogenes gpsB mutants are unable to grow at 42°C, but we observed that spontaneous suppressors correcting this defect arise on agar plates with high frequency. We here describe a first set of gpsB suppressors that mapped to the clpC and murZ genes. While ClpC is the ATPase component of the Clp protease, MurZ is a paralogue of the listerial UDP-N-acetylglucosamine (UDP-GlcNAc) 1-carboxyvinyltransferase MurA. Both enzymes catalyze the enolpyruvyl transfer from phosphoenolpyruvate to UDP-GlcNAc, representing the first committed step of peptidoglycan biosynthesis. We confirmed that clean deletion of the clpC or murZ gene suppressed the ΔgpsB phenotype. It turned out that the absence of either gene leads to accumulation of MurA, and we show that artificial overexpression of MurA alone was sufficient for suppression. Inactivation of other UDP-GlcNAc-consuming pathways also suppressed the heat-sensitive growth of the ΔgpsB mutant, suggesting that an increased influx of precursor molecules into peptidoglycan biosynthesis can compensate for the lack of GpsB. Our results support a model according to which PBP A1 becomes misregulated and thus toxic in the absence of GpsB due to unproductive consumption of cell wall precursor molecules. The late cell division protein GpsB is important for cell wall biosynthesis in Gram-positive bacteria. GpsB of the human pathogen L. monocytogenes interacts with one of the key enzymes of this pathway, penicillin binding protein A1 (PBP A1), and influences its activity. PBP A1 catalyzes the last two steps of cell wall biosynthesis, but it is unknown how GpsB controls PBP A1. We observed that a L. monocytogenes gpsB mutant forms spontaneous suppressors and have mapped their mutations to genes mediating and influencing the first step of cell wall biosynthesis, likely stimulating the influx of metabolites into this pathway. We assume that GpsB is important to ensure productive incorporation of cell wall precursors into the peptidoglycan sacculus by PBP A1.

Dataset Information

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

Publications

Metabolic Engineering of <i>Bacillus subtilis</i> Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production.

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