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Metabolic engineering of Bacillus subtilis for terpenoid production.

ABSTRACT: Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired "cell factory" to produce terpenoids.

SUBMITTER: Guan Z

PROVIDER: S-EPMC4628092 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Metabolic engineering of Bacillus subtilis for terpenoid production.

Guan Zheng Z Xue Dan D Abdallah Ingy I II Dijkshoorn Linda L Setroikromo Rita R Lv Guiyuan G Quax Wim J WJ

Applied microbiology and biotechnology 20150915 22

Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are bot ...[more]

PMID: 26373726

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Project description: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.

Project description:BackgroundFlux Balance Analysis (FBA) is a well-known bioinformatics tool for metabolic engineering design. Previously, we have successfully used single-level FBA to design metabolic fluxes in Bacillus subtilis to enhance (R,R)-2,3-butanediol (2,3-BD) production from glycerol. OptKnock is another powerful technique for devising gene deletion strategies to maximize microbial growth coupling with improved biochemical production. It has never been used in B. subtilis. In this study, we aimed to compare the use of single-level FBA and OptKnock for designing enhanced 2,3-BD production from glycerol in B. subtilis.ResultsSingle-level FBA and OptKnock were used to design metabolic engineering approaches for B. subtilis to enhance 2,3-BD production from glycerol. Single-level FBA indicated that deletion of ackA, pta, lctE, and mmgA would improve the production of 2,3-BD from glycerol, while OptKnock simulation suggested the deletion of ackA, pta, mmgA, and zwf. Consequently, strains LM01 (single-level FBA-based) and MZ02 (OptKnock-based) were constructed, and their capacity to produce 2,3-BD from glycerol was investigated. The deletion of multiple genes did not negatively affect strain growth and glycerol utilization. The highest 2,3-BD production was detected in strain LM01. Strain MZ02 produced 2,3-BD at a similar level as the wild type, indicating that the OptKnock prediction was erroneous. Two-step FBA was performed to examine the reason for the erroneous OptKnock prediction. Interestingly, we newly found that zwf gene deletion in strain MZ02 improved lactate production, which has never been reported to date. The predictions of single-level FBA for strain MZ02 were in line with experimental findings.ConclusionsWe showed that single-level FBA is an effective approach for metabolic design and manipulation to enhance 2,3-BD production from glycerol in B. subtilis. Further, while this approach predicted the phenotypes of generated strains with high precision, OptKnock prediction was not accurate. We suggest that OptKnock modelling predictions be evaluated by using single-level FBA to ensure the accuracy of metabolic pathway design. Furthermore, the zwf gene knockout resulted in the change of metabolic fluxes to enhance the lactate productivity.

Dataset Information

Metabolic engineering of Bacillus subtilis for terpenoid production.

Publications

Metabolic engineering of Bacillus subtilis for terpenoid production.

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