Dataset Information

Production of Squalene in Bacillus subtilis by Squalene Synthase Screening and Metabolic Engineering.

ABSTRACT: Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in Bacillus subtilis and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS candidates from various organisms, comparison of expression vectors, optimization of cultivation temperatures, and examination of rate-limiting factors within the synthetic pathway. We were, for the first time, able to obtain squalene synthesis in B. subtilis. Furthermore, we achieved a 29-fold increase of squalene yield (0.26-7.5 mg/L) by expressing SQS from Bacillus megaterium and eliminating bottlenecks within the upstream methylerythritol-phosphate pathway. Moreover, our findings showed that also ispA could positively affect the production of squalene.

SUBMITTER: Song Y

PROVIDER: S-EPMC7168599 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Production of Squalene in <i>Bacillus subtilis</i> by Squalene Synthase Screening and Metabolic Engineering.

Song Yafeng Y Guan Zheng Z van Merkerk Ronald R Pramastya Hegar H Abdallah Ingy I II Setroikromo Rita R Quax Wim J WJ

Journal of agricultural and food chemistry 20200403 15

Squalene synthase (SQS) catalyzes the conversion of two farnesyl pyrophosphates to squalene, an important intermediate in between isoprene and valuable triterpenoids. In this study, we have constructed a novel biosynthesis pathway for squalene in <i>Bacillus subtilis</i> and performed metabolic engineering aiming at facilitating further exploitation and production of squalene-derived triterpenoids. Therefore, systematic studies and analysis were performed including selection of multiple SQS cand ...[more]

PMID: 32208656

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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

Production of Squalene in Bacillus subtilis by Squalene Synthase Screening and Metabolic Engineering.

Publications

Production of Squalene in <i>Bacillus subtilis</i> by Squalene Synthase Screening and Metabolic Engineering.

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