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Yıl 2022, Cilt: 26 Sayı: 6, 1656 - 1664, 28.06.2025

Tresna Lestari Catur Riani Elfahmi Elfahmi , Asep Gana Suganda

https://doi.org/10.29228/jrp.256

Öz

Kaynakça

  • [1] WHO World malaria report 2021. https://www.who.int/teams/global-malaria-programme/reports/worldmalaria-report-2021 (accessed on 15 December 2021).
  • [2] Tu Y. Artemisinin - A gift from traditional Chinese medicine to the world (Nobel Lecture). Angew Chem Int Ed Engl. 2016; 55(35): 10210-10226. [CrossRef]
  • [3] Numonov S, Farukh S, Aminjon S, Parviz S, Sunbula A, Ramazon S, Maidina H. Assessment of artemisinin contents in selected Artemisia species from Tajikistan (Central Asia). Medicines (Basel). 2019; 6(1): 23. [CrossRef]
  • [4] Czechowski T, Larson TR, Catania TM, Harvey D, Wei C, Essome M, Brown GD, Graham IA. Detailed phytochemical analysis of high and low artemisinin-producing chemotypes of Artemisia annua. Front Plant Sci. 2018; 9: 641. [CrossRef]
  • [5] Nguyen KT, Arsenault PR, Weathers PJ. Trichomes + roots + ROS = artemisinin: regulating artemisinin biosynthesis in Artemisia annua L. In Vitro Cell Dev Biol Plant. 2011; 47(3): 329–338. [CrossRef]
  • [6] Wen W, Yu R. Artemisinin biosynthesis and its regulatory enzymes: Progress and perspective. Phcog Rev. 2011; 5(10): 189–194. [CrossRef]
  • [7] Pateraki I, Heskes AM, Hamberger B. Cytochromes P450 for terpene functionalisation and metabolic engineering. Adv Biochem Eng Biotechnol. 2015; 148: 107-139. [CrossRef]
  • [8] Yang C, Gao X, Jiang Y, Sun B, Gao F, Yang S. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli. Metab Eng. 2016; 37: 79-91. [CrossRef]
  • [9] Zhao Y, Yang J, Qin B, Li Y. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway. Appl Microbiol Biotechnol. 2011; 90: 1915–1922. [CrossRef]
  • [10] Zhou K, Zou R, Zhang C, Stephanopoulos G, Too H. Optimization of amorphadiene synthesis in Bacillus subtilis via transcriptional, translational and media modulation. Biotechnol Bioeng. 2013; 110(9): 2556–2561. [CrossRef]
  • [11] Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003; 21(7): 796–802. [CrossRef]
  • [12] Newman JD, Marshall J, Chang M, Nowroozi F, Paradise E, Pitera D, Newman KL, Keasling JD. High-Level Production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol Bioeng. 2006; 95(4): 684-691. [CrossRef]
  • [13] Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, Anthony LC, Regentin R, Keasling JD, Renninger NS, Newman JD. High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One. 2009; 4(2): e4489. [CrossRef]
  • [14] Wang J, Apptec W, Xiong Z. Engineering MEP Pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control. Chin J Biotech. 2014; 30(1): 64-75. [CrossRef]
  • [15] Chung BK, Klement M, Lakshmanan M, Mohanty B, Lee D. Genome-scale in silico modeling and analysis for designing synthetic terpenoid-producing microbial cell factories. Chem Eng Sci. 2012; 103(September): 1–9. [CrossRef]
  • [16] Wang Q, Quan S, Xiao H. Towards efficient terpenoid biosynthesis: manipulating IPP and DMAPP supply. Bioresour Bioprocess. 2019; 6(6): 1-13. [CrossRef]
  • [17] Yuan LZ, Rouvière PE, LaRossa RA, Suh W. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab Eng. 2006; 8(1): 79–90. [CrossRef]
  • [18] Volke DC, Rohwer J, Fischer R, Jennewein S. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microb Cell Fact. 2019; 18(1): 192. [CrossRef]
  • [19] Kane JF. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol. 1995; 6(5): 494–500. [CrossRef]
  • [20] Kurland C, Gallantt J. Errors of heterologous protein expression. Curr Opin Biotechnol. 1996; 7(5): 489-493. [CrossRef]
  • [21] Gustafsson C, Minshull J, Govindarajan S, Ness J, Villalobos A, Welch M, Drive OB, Suite A, Park M. Engineering genes for predictable protein expression. Protein Expr Purif. 2012; 83(1): 37–46. [CrossRef]
  • [22] Wallaart TE, Bouwmeester HJ, Hille J, Poppinga L, Maijers NCA. Amorpha-4,11-diene synthase: Cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta. 2001; 212(3): 460–465. [CrossRef]
  • [23] Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Mai A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Jiang H, Fleck M, Bajad S, Dang G, … Newman JD. High-level semisynthetic production of the potent antimalarial artemisinin. Nature. 2013; 496(7446): 528–532. [CrossRef]
  • [24] Peplow M. Synthetic malaria drug meets market resistance. Nature. 2016; 530: 389–390. [CrossRef]
  • [25] Feth MP, Rossen K, Burgard A. A Pilot Plant PAT Approach for the Diastereoselective Diimide Reduction of Artemisinic Acid. Org Process Res Dev. 2013; 17(2): 282–293. [CrossRef]
  • [26] Amara Z, Bellamy JFB, Horvath R, Miller SJ, Beeby A, Burgard A, Rossen K, Poliakoff M, George MW. Applying green chemistry to the photochemical route to artemisinin. Nat Chem. 2015; 7: 489-495. [CrossRef]
  • [27] Gilmore K, Daniel K, Ju WL, Zoltán, McQuade DT, Seidel-Morgenstern A, Seeberger PH. Continuous synthesis of artemisinin-derived medicines. Chem Commun (Camb). 2014; 50(84): 12652-12655. [CrossRef]
  • [28] Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671-675. [CrossRef]
  • [29] Schein CH. Production of soluble recombinant proteins in bacteria. Nat Biotechno. 1989; 7: 1141–1149. [CrossRef]
  • [30] Feller G, Bussy OLE. Expression of psychrophilic genes in mesophilic hosts: Assessment of the folding state of a recombinant alpha-amylase. Appl Environ Microbiol. 1998; 64(3): 1163–1165. [CrossRef]
  • [31] Vallejo LF, Rinas U. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact. 2004; 3(1): 11. [CrossRef]
  • [32] Chung CT, Niemela SL, Miller RH. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA. 1989; 86(7): 2172–2175. [CrossRef]

Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli

Yıl 2022, Cilt: 26 Sayı: 6, 1656 - 1664, 28.06.2025

Tresna Lestari Catur Riani Elfahmi Elfahmi , Asep Gana Suganda

https://doi.org/10.29228/jrp.256

Öz

Artemisinin combination therapies (ACTs) have become the mainstay of treatment for malaria worldwide. This has led to a high demand for artemisinin precursors as starting materials for artemisinin production and their semisynthetic derivatives. In this study, heterologous expression of enzymes involved in artemisinin biosynthesis was performed in Escherichia coli to produce artemisinin precursors, i.e., amorphadiene, artemisinic acid, and dihydroartemisinic acid. These enzymes are farnesyl pyrophosphate synthase (FPS), amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1/CYP), artemisinic aldehyde delta-11(13) reductase (DBR2), and aldehyde dehydrogenase 1 (ALDH1). Overexpression of the heterologous 1-deoxy-D-xylulose 5-phosphate (DXP) synthase gene (dxs) from Bacillus subtilis and the native isopentenyl diphosphate delta isomerase (IDI) gene (idi) from E. coli was also performed to enhance isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via the methylerithritol phosphate (MEP) pathway in E. coli. All genes were cloned into three plasmids. Gene expression was performed under isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction and characterized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The number of IPTG, incubation temperature, length of incubation time, and culture medium were optimized to find the best conditions for protein expression. It is found that all proteins could be expressed under 0.5 mM IPTG induction at an incubation temperature of 20 °C for 24 hours in Luria-Bertani medium. The cloning of seven key enzymes involved in artemisinin biosynthesis in E. coli has never been described in other studies. By further optimizing the fermentation process, this research offers a promising future for the production of artemisinin precursors in E. coli.

Anahtar Kelimeler

heterologous expression MEP pathway artemisinin Escherichia coli

Kaynakça

  • [1] WHO World malaria report 2021. https://www.who.int/teams/global-malaria-programme/reports/worldmalaria-report-2021 (accessed on 15 December 2021).
  • [2] Tu Y. Artemisinin - A gift from traditional Chinese medicine to the world (Nobel Lecture). Angew Chem Int Ed Engl. 2016; 55(35): 10210-10226. [CrossRef]
  • [3] Numonov S, Farukh S, Aminjon S, Parviz S, Sunbula A, Ramazon S, Maidina H. Assessment of artemisinin contents in selected Artemisia species from Tajikistan (Central Asia). Medicines (Basel). 2019; 6(1): 23. [CrossRef]
  • [4] Czechowski T, Larson TR, Catania TM, Harvey D, Wei C, Essome M, Brown GD, Graham IA. Detailed phytochemical analysis of high and low artemisinin-producing chemotypes of Artemisia annua. Front Plant Sci. 2018; 9: 641. [CrossRef]
  • [5] Nguyen KT, Arsenault PR, Weathers PJ. Trichomes + roots + ROS = artemisinin: regulating artemisinin biosynthesis in Artemisia annua L. In Vitro Cell Dev Biol Plant. 2011; 47(3): 329–338. [CrossRef]
  • [6] Wen W, Yu R. Artemisinin biosynthesis and its regulatory enzymes: Progress and perspective. Phcog Rev. 2011; 5(10): 189–194. [CrossRef]
  • [7] Pateraki I, Heskes AM, Hamberger B. Cytochromes P450 for terpene functionalisation and metabolic engineering. Adv Biochem Eng Biotechnol. 2015; 148: 107-139. [CrossRef]
  • [8] Yang C, Gao X, Jiang Y, Sun B, Gao F, Yang S. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli. Metab Eng. 2016; 37: 79-91. [CrossRef]
  • [9] Zhao Y, Yang J, Qin B, Li Y. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway. Appl Microbiol Biotechnol. 2011; 90: 1915–1922. [CrossRef]
  • [10] Zhou K, Zou R, Zhang C, Stephanopoulos G, Too H. Optimization of amorphadiene synthesis in Bacillus subtilis via transcriptional, translational and media modulation. Biotechnol Bioeng. 2013; 110(9): 2556–2561. [CrossRef]
  • [11] Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003; 21(7): 796–802. [CrossRef]
  • [12] Newman JD, Marshall J, Chang M, Nowroozi F, Paradise E, Pitera D, Newman KL, Keasling JD. High-Level Production of amorpha-4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotechnol Bioeng. 2006; 95(4): 684-691. [CrossRef]
  • [13] Tsuruta H, Paddon CJ, Eng D, Lenihan JR, Horning T, Anthony LC, Regentin R, Keasling JD, Renninger NS, Newman JD. High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli. PLoS One. 2009; 4(2): e4489. [CrossRef]
  • [14] Wang J, Apptec W, Xiong Z. Engineering MEP Pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control. Chin J Biotech. 2014; 30(1): 64-75. [CrossRef]
  • [15] Chung BK, Klement M, Lakshmanan M, Mohanty B, Lee D. Genome-scale in silico modeling and analysis for designing synthetic terpenoid-producing microbial cell factories. Chem Eng Sci. 2012; 103(September): 1–9. [CrossRef]
  • [16] Wang Q, Quan S, Xiao H. Towards efficient terpenoid biosynthesis: manipulating IPP and DMAPP supply. Bioresour Bioprocess. 2019; 6(6): 1-13. [CrossRef]
  • [17] Yuan LZ, Rouvière PE, LaRossa RA, Suh W. Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. Metab Eng. 2006; 8(1): 79–90. [CrossRef]
  • [18] Volke DC, Rohwer J, Fischer R, Jennewein S. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microb Cell Fact. 2019; 18(1): 192. [CrossRef]
  • [19] Kane JF. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol. 1995; 6(5): 494–500. [CrossRef]
  • [20] Kurland C, Gallantt J. Errors of heterologous protein expression. Curr Opin Biotechnol. 1996; 7(5): 489-493. [CrossRef]
  • [21] Gustafsson C, Minshull J, Govindarajan S, Ness J, Villalobos A, Welch M, Drive OB, Suite A, Park M. Engineering genes for predictable protein expression. Protein Expr Purif. 2012; 83(1): 37–46. [CrossRef]
  • [22] Wallaart TE, Bouwmeester HJ, Hille J, Poppinga L, Maijers NCA. Amorpha-4,11-diene synthase: Cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta. 2001; 212(3): 460–465. [CrossRef]
  • [23] Paddon CJ, Westfall PJ, Pitera DJ, Benjamin K, Fisher K, McPhee D, Leavell MD, Tai A, Mai A, Eng D, Polichuk DR, Teoh KH, Reed DW, Treynor T, Lenihan J, Jiang H, Fleck M, Bajad S, Dang G, … Newman JD. High-level semisynthetic production of the potent antimalarial artemisinin. Nature. 2013; 496(7446): 528–532. [CrossRef]
  • [24] Peplow M. Synthetic malaria drug meets market resistance. Nature. 2016; 530: 389–390. [CrossRef]
  • [25] Feth MP, Rossen K, Burgard A. A Pilot Plant PAT Approach for the Diastereoselective Diimide Reduction of Artemisinic Acid. Org Process Res Dev. 2013; 17(2): 282–293. [CrossRef]
  • [26] Amara Z, Bellamy JFB, Horvath R, Miller SJ, Beeby A, Burgard A, Rossen K, Poliakoff M, George MW. Applying green chemistry to the photochemical route to artemisinin. Nat Chem. 2015; 7: 489-495. [CrossRef]
  • [27] Gilmore K, Daniel K, Ju WL, Zoltán, McQuade DT, Seidel-Morgenstern A, Seeberger PH. Continuous synthesis of artemisinin-derived medicines. Chem Commun (Camb). 2014; 50(84): 12652-12655. [CrossRef]
  • [28] Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012; 9(7): 671-675. [CrossRef]
  • [29] Schein CH. Production of soluble recombinant proteins in bacteria. Nat Biotechno. 1989; 7: 1141–1149. [CrossRef]
  • [30] Feller G, Bussy OLE. Expression of psychrophilic genes in mesophilic hosts: Assessment of the folding state of a recombinant alpha-amylase. Appl Environ Microbiol. 1998; 64(3): 1163–1165. [CrossRef]
  • [31] Vallejo LF, Rinas U. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact. 2004; 3(1): 11. [CrossRef]
  • [32] Chung CT, Niemela SL, Miller RH. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci USA. 1989; 86(7): 2172–2175. [CrossRef]
Toplam 32 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Farmasotik Biyoteknoloji
Bölüm Articles
Yazarlar

Tresna Lestari

Catur Riani

Elfahmi Elfahmi

Asep Gana Suganda

Yayımlanma Tarihi 28 Haziran 2025
Yayımlandığı Sayı Yıl 2022 Cilt: 26 Sayı: 6

Kaynak Göster

APA Lestari, T., Riani, C., Elfahmi, E., Suganda, A. G. (2025). Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli. Journal of Research in Pharmacy, 26(6), 1656-1664. https://doi.org/10.29228/jrp.256
AMA Lestari T, Riani C, Elfahmi E, Suganda AG. Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli. J. Res. Pharm. Haziran 2025;26(6):1656-1664. doi:10.29228/jrp.256
Chicago Lestari, Tresna, Catur Riani, Elfahmi Elfahmi, ve Asep Gana Suganda. “Heterologous Expression of Enzymes Involved in Artemisinin Biosynthesis via Methylerythritol Phosphate Pathway from Artemisia Annua in Escherichia Coli”. Journal of Research in Pharmacy 26, sy. 6 (Haziran 2025): 1656-64. https://doi.org/10.29228/jrp.256.
EndNote Lestari T, Riani C, Elfahmi E, Suganda AG (01 Haziran 2025) Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli. Journal of Research in Pharmacy 26 6 1656–1664.
IEEE T. Lestari, C. Riani, E. Elfahmi, ve A. G. Suganda, “Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli”, J. Res. Pharm., c. 26, sy. 6, ss. 1656–1664, 2025, doi: 10.29228/jrp.256.
ISNAD Lestari, Tresna vd. “Heterologous Expression of Enzymes Involved in Artemisinin Biosynthesis via Methylerythritol Phosphate Pathway from Artemisia Annua in Escherichia Coli”. Journal of Research in Pharmacy 26/6 (Haziran 2025), 1656-1664. https://doi.org/10.29228/jrp.256.
JAMA Lestari T, Riani C, Elfahmi E, Suganda AG. Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli. J. Res. Pharm. 2025;26:1656–1664.
MLA Lestari, Tresna vd. “Heterologous Expression of Enzymes Involved in Artemisinin Biosynthesis via Methylerythritol Phosphate Pathway from Artemisia Annua in Escherichia Coli”. Journal of Research in Pharmacy, c. 26, sy. 6, 2025, ss. 1656-64, doi:10.29228/jrp.256.
Vancouver Lestari T, Riani C, Elfahmi E, Suganda AG. Heterologous expression of enzymes involved in artemisinin biosynthesis via methylerythritol phosphate pathway from Artemisia annua in Escherichia coli. J. Res. Pharm. 2025;26(6):1656-64.