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Metabolic Engineering of Bacillus Subtilis Toward Taxadiene View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Groningen University of Groningen Metabolic engineering of bacillus subtilis toward taxadiene biosynthesis as the first committed step for taxol production Abdallah, Ingy I.; Pramastya, Hegar; Van Merkerk, Ronald; Sukrasno; Quax, Wim J. Published in: Frontiers in Microbiology DOI: 10.3389/fmicb.2019.00218 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Abdallah, I. I., Pramastya, H., Van Merkerk, R., Sukrasno, & Quax, W. J. (2019). Metabolic engineering of bacillus subtilis toward taxadiene biosynthesis as the first committed step for taxol production. Frontiers in Microbiology, 10(FEB), [218]. https://doi.org/10.3389/fmicb.2019.00218 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 13-11-2019 ORIGINAL RESEARCH published: 20 February 2019 doi: 10.3389/fmicb.2019.00218 Metabolic Engineering of Bacillus subtilis Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production Ingy I. Abdallah1†, Hegar Pramastya1,2†, Ronald van Merkerk1, Sukrasno2 and Wim J. Quax1* 1 Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen, The Netherlands, 2 Pharmaceutical Biology Research Group, School of Pharmacy, Institut Teknologi Bandung, Bandung, Indonesia Edited by: José E. Barboza-Corona, Terpenoids are natural products known for their medicinal and commercial applications. Universidad de Guanajuato, Metabolic engineering of microbial hosts for the production of valuable compounds, such Mexico as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Reviewed by: Regarded As Safe (GRAS) Bacillus subtilis 168 with its broad metabolic potential is Shawn Chen, Global Health Drug Discovery considered one of these interesting microbial hosts. In the effort toward engineering B. Institute, Tsinghua University, China subtilis as a cell factory for the production of the chemotherapeutic Taxol, we expressed Hwan You Chang, National Tsing Hua University, Taiwan the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion *Correspondence: of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the Wim J. Quax first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight [email protected] enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP † These authors have contributed precursor. This was achieved by creating a synthetic operon harboring the B. subtilis equally to this work genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (dxs, ispD, ispF, Specialty section: ispH, ispC, ispE, ispG) together with ispA (encoding geranyl and farnesyl pyrophosphate This article was submitted to synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector Microbiotechnology, Ecotoxicology and Bioremediation, harboring the crtE gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of a section of the journal Pantoea ananatis) to increase the supply of GGPP was introduced. The overexpression Frontiers in Microbiology of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase Received: 02 November 2018 Accepted: 28 January 2019 in the amount of taxadiene produced compared to the strain only expressing TXS and Published: 20 February 2019 relying on the innate pathway of B. subtilis. The total amount of taxadiene produced by Citation: that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene Abdallah II, Pramastya H, synthase in B. subtilis. We determined that the expression of GGPPS through the crtE van Merkerk R, Sukrasno and Quax WJ (2019) Metabolic gene is essential for the formation of sufficient precursor, GGPP, inB. subtilis as its innate Engineering of Bacillus subtilis metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene Toward Taxadiene Biosynthesis as the First Committed Step for production by overexpressing the complete MEP pathway along with IspA and GGPPS Taxol Production. presents the prospect for further engineering aiming for semisynthesis of Taxol. Front. Microbiol. 10:218. doi: 10.3389/fmicb.2019.00218 Keywords: Bacillus subtilis, metabolite, MEP, GGPPS, taxadiene, Taxol Frontiers in Microbiology | www.frontiersin.org 1 February 2019 | Volume 10 | Article 218 Abdallah et al. Engineering Bacillus subtilis for Taxadiene Biosynthesis INTRODUCTION for their medicinal value or use as flavors and fragrances. Among the most famous medicinally important terpenoids are the Terpenoids represent the largest, structurally and functionally antimalarial artemisinin from the plant Artemisia annua and most varied group of natural products. This diversity is based the anticancer paclitaxel (Taxol®) from the yew trees (Taxus on a structural complexity that cannot be simply reproduced brevifolia or Taxus baccata). The majority of terpenoids are using chemical synthetic processes. Nowadays, there are over naturally produced in low amounts and their extraction is usually 50,000 known terpenoids, a lot of which are biosynthesized by labor intensive, and it entails considerable consumption of natural plants. Numerous terpenoids have attracted commercial interest resources. For instance, the production of enough Taxol® to A B FIGURE 1 | (A) Biosynthesis of taxa-4,11-diene via the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in Bacillus subtilis. Intermediates in the metabolic pathway: 1-deoxy-D-xylulose 5-phosphate (DXP), 2-C-methyl-D-erythritol 4-phosphate (MEP), 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-ME), 2-phospho-4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol (CDP-MEP), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC), (E)-4-hydroxy-3-methylbut-2-en-1- yl diphosphate (HMBPP), isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). Enzymes in the biosynthesis pathway: 1-deoxy-D-xylulose-5-phosphate synthase (Dxs), 1-deoxy-D-xylulose-5- phosphate reductoisomerase, or 2-C-methyl-D-erythritol 4-phosphate synthase (Dxr, IspC), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD), 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), (E)-4-hydroxy-3-methylbut-2-enyl- diphosphate synthase (IspG), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH), isopentenyldiphosphate delta-isomerase (Idi), IspA which act as geranyl pyrophosphate synthase (GPPS) and farnesyl pyrophosphate synthase (FPPS), geranylgeranyl pyrophosphate synthase (GGPPS), and taxadiene synthase (TXS). (B) Plasmid constructs used for engineering B. subtilis. pDR_txs contains txs gene (red), preceded with B. subtilis mntA ribosomal binding site (dark red), to be inserted into the genome of B. subtilis between the amyE front flanking region and amyE back flanking region (purple), IPTG inducible hyperspank promoter (pink), and ampicillin and spectinomycin resistance cassettes (green). pBS0E_crtE contains crtE gene (yellow) encoding for GGPPS and preceded with B. subtilis mntA ribosomal binding site (dark red), xylose inducible promoter (orange), and ampicillin and erythromycin resistance cassettes (green). p04_SDFHCEGA contains seven genes of the MEP pathway, dxs, ispD, ispF, ispH, ispC, ispE, and ispG, along with the gene ispA (blue), each preceded with B. subtilis mntA ribosomal binding site (dark red), in a synthetic operon controlled by xylose inducible promoter (orange) and ampicillin and chloramphenicol resistance cassettes (green). Frontiers in Microbiology | www.frontiersin.org 2 February 2019 | Volume 10 | Article 218 Abdallah et al. Engineering Bacillus subtilis for Taxadiene Biosynthesis treat one cancer patient would approximately require six 100-year- et al., 2010), Saccharomyces cerevisiae (DeJong et al., 2006; old Pacific yew trees, and similarly, there are reports of enormous Engels et al., 2008), and the transgenic plant Arabidopsis shortfalls in artemisinin production due to seed shortage. In thaliana (Besumbes et al., 2004). Based on the success of addition, chemical synthesis and modification of most terpenoids taxadiene production in these hosts, Bacillus subtilis represents is tremendously difficult and problematic because of the complexity an interesting microbial host for the production of taxadiene and chirality of their chemical structures. Hence, researchers where it has
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