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Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

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Abstract

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.

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Acknowledgments

We would like to acknowledge the financial support from Singapore-MIT Alliance.

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Authors and Affiliations

  1. Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore, Singapore

    Congqiang Zhang, Ruiyang Zou, Xixian Chen, Gregory Stephanopoulos & Heng-Phon Too

  2. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Blk MD7, #05-04, Singapore, 117597, Singapore

    Congqiang Zhang, Xixian Chen & Heng-Phon Too

  3. Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, USA

    Gregory Stephanopoulos

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  1. Congqiang Zhang
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  2. Ruiyang Zou
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  5. Heng-Phon Too
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Correspondence to Heng-Phon Too.

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Zhang, C., Zou, R., Chen, X. et al. Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production. Appl Microbiol Biotechnol 99, 3825–3837 (2015). https://doi.org/10.1007/s00253-015-6463-y

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  • DOI: https://doi.org/10.1007/s00253-015-6463-y

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