Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells

Malhotra, Karan; Subramaniyan, Mayavan; Rawat, Khushboo; Kalamuddin, Md.; Qureshi, M. Irfan; Malhotra, Pawan; Mohmmed, Asif; Cornish, Katrina; Daniell, Henry; Kumar, Shashi

doi:10.1016/j.molp.2016.09.013

Cited by 93 publications

(64 citation statements)

References 74 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, we monitored the formation of the intermediates IP, IPP/DMAPP, GPP, FPP, and GGPP using LC‐MS/MS (Figure S2B). All of the expected products were identified by their electron ionization mass spectra using purchased standards (valencene and limonene), a standard purified in‐house (taxadiene), or from the literature (amorphadiene; Malhotra et al, ) and the MS spectra of the formed products are shown in Figure S2C–F. At the concentrations used, what we suspect was protein precipitation was visible after 24 hr, but was prevented by the addition of 0.025% Tween 20 (w/v; data not shown) in subsequent experiments.…”

Section: Resultsmentioning

confidence: 96%

Cell free biosynthesis of isoprenoids from isopentenol

Ward

Chatzivasileiou

Stephanopoulos

2019

Biotech & Bioengineering

View full text Add to dashboard Cite

Cell-free systems are growing in importance for the biosynthesis of complex molecules. These systems combine the precision of traditional chemistry with the versatility of biology in creating superior overall processes. Recently, a new synthetic pathway for the biosynthesis of isoprenoids using the substrate isopentenol, dubbed the isopentenol utilization pathway (IUP), was demonstrated to be a promising alternative to the native 2C-methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathways. This simplified pathway, which contains a minimum of four enzymes to produce basic monoterpenes and only depends on ATP and isopentenol as substrates, allows for a highly flexible approach to the commercial synthesis of isoprenoid products. In this work, we use metabolic reconstitution to characterize this new pathway in vitro and demonstrate its use for the cell-free synthesis of mono-, sesquit-, and diterpenoids. Kinetic modeling and sensitivity analysis were also used to identify the most significant parameters for taxadiene productivity, and metabolic control analysis was employed to elucidate protein-level interactions within this pathway, which demonstrated that the IUP enzymatic system is primarily controlled by the concentration and kinetics of choline kinase (CK) and not regulated by any pathway intermediates. This is a significant advantage over the natural MEP or MVA pathways as it greatly simplifies future metabolic engineering efforts, both in vitro and in vivo, aiming at improving the kinetics of CK. Finally, we used the insights gathered to demonstrate an in vitro IUP system that can produce 220 mg/L of the diterpene taxadiene, in 9 hr, almost 3-fold faster than any system reported thus far. K E Y W O R D Sbiocatalysis, enzyme kinetics, isoprenoids, metabolic control analysis, taxadiene

show abstract

Section: Resultsmentioning

confidence: 96%

Cell free biosynthesis of isoprenoids from isopentenol

Ward

Chatzivasileiou

Stephanopoulos

2019

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…This may be one of the reasons for the failure to obtain transgenic A. annua lines with high artemisinin content. Recently, a synthetic biology strategy for artemisinin biosynthesis, in which the complete biosynthetic pathway of artemisinic acid, the precursor of artemisinin, is introduced into tobacco plants, has also been reported (Fuentes et al, 2016;Malhotra et al, 2016). However, whether this strategy allows efficient artemisinin production in other plant species needs to be further studied.…”

Section: Introductionmentioning

confidence: 99%

The Genome of Artemisia annua Provides Insight into the Evolution of Asteraceae Family and Artemisinin Biosynthesis

et al. 2018

View full text Add to dashboard Cite

Artemisia annua, commonly known as sweet wormwood or Qinghao, is a shrub native to China and has long been used for medicinal purposes. A. annua is now cultivated globally as the only natural source of a potent anti-malarial compound, artemisinin. Here, we report a high-quality draft assembly of the 1.74-gigabase genome of A. annua, which is highly heterozygous, rich in repetitive sequences, and contains 63 226 protein-coding genes, one of the largest numbers among the sequenced plant species. We found that, as one of a few sequenced genomes in the Asteraceae, the A. annua genome contains a large number of genes specific to this large angiosperm clade. Notably, the expansion and functional diversification of genes encoding enzymes involved in terpene biosynthesis are consistent with the evolution of the artemisinin biosynthetic pathway. We further revealed by transcriptome profiling that A. annua has evolved the sophisticated transcriptional regulatory networks underlying artemisinin biosynthesis. Based on comprehensive genomic and transcriptomic analyses we generated transgenic A. annua lines producing high levels of artemisinin, which are now ready for large-scale production and thereby will help meet the challenge of increasing global demand of artemisinin.

show abstract

“…The most successful attempt to biosynthetically produce artemisinin took two large sections of the metabolic pathway for artemisinin and genetically engineered them separately into three different cellular compartments (chloroplast, nucleus, and mitochondria). The resulting heterologous expression of artemisinin at ∼0.8 mg/g DW was less than in the native plant, which can reach 31.4 mg/g DW (Zhang et al, 2009;Malhotra et al, 2016). This can in part be explained by the complexity of the gene expression and regulation of the biosynthetic pathway (Ikram and Simonsen, 2017).…”

Section: Molecular Farmingmentioning

confidence: 98%