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Chapter 25 : Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug

Authors: Christopher J. Petzold, Jay D. Keasling
Content Type: Reference
Publication Year: 2010

Category: Applied and Industrial Microbiology

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Abstract:

Biotechnology offers multiple means for improving artemisinin production. Synthetic biology and metabolic engineering of microbes offer an attractive alternative to natural sources as a means for inexpensive pharmaceutical production. The isoprenoid precursors used in artemisinin biosynthesis are common to both prokaryotes and eukaryotes; consequently, model microbes, such as and , can be used to convert simple, cheap sugar to artemisinin precursors. Optimal metabolite analysis depends on the detection of all engineered pathway intermediates, the carbon source, and the common metabolites. Chromatography, both gas and liquid, and capillary electrophoresis were the main techniques used to separate the metabolites of interest from the rest of the metabolites present. In general, these methods were coupled to mass spectrometry (MS) for identification. Mutagenic and combinatorial methods are powerful tools for optimizing metabolically engineered systems. Optimal pathway gene expression is dependent on a wide variety of factors including, but not limited to, gene product toxicity, solubility, codon usage, mRNA secondary structure, mRNA stability, and translational efficiency. Protein engineering of P450 BM-3 yielded selective oxidation of amorpha-4,11-diene at a high rate to produce artemisinic epoxide at titers of 250 mg/liter.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25

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Environmental Microbiology
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Acetyl Coenzyme A
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Fatty Acid Biosynthesis
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Metabolic Engineering of Escherichia coli for the Production of a Precursor to Artemisinin, an Antimalarial Drug
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Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
/content/10.1128/9781555816827.ch25.ch25fig01
FIGURE 1
Biosynthetic routes to polyprenyl pyrophosphate isoprenoid biosynthetic pathways in and . Dxs, DXP synthase; IspC, DXP reductoisomerase; IspD, 4-diphosphocytidyl-2--methyl--erythritol synthase; IspE, 4-diphosphocytidyl-2--methyl--erythritol kinase; IspF, 2--methyl--erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-()-butenyl 4-diphosphate synthase; IspH, 1-hydroxy-2-methyl-2-()-butenyl 4-diphosphate reductase; AtoB, acetoacetyl-CoA thiolase; HMGS, 3-hydroxymethylglutaryl-CoA synthase; HMGR, 3-hydroxymethylglutaryl-CoA reductase 1; MK, mevalonate kinase; PMK, phospho-mevalonate kinase; MPD, mevalonate pyrophosphate decarboxylase; Idi, isopentenyl pyrophosphate isomerase. Adapted from ( ) with permission of the publisher.
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FIGURE 1

Biosynthetic routes to polyprenyl pyrophosphate isoprenoid biosynthetic pathways in and . Dxs, DXP synthase; IspC, DXP reductoisomerase; IspD, 4-diphosphocytidyl-2--methyl--erythritol synthase; IspE, 4-diphosphocytidyl-2--methyl--erythritol kinase; IspF, 2--methyl--erythritol 2,4-cyclodiphosphate synthase; IspG, 1-hydroxy-2-methyl-2-()-butenyl 4-diphosphate synthase; IspH, 1-hydroxy-2-methyl-2-()-butenyl 4-diphosphate reductase; AtoB, acetoacetyl-CoA thiolase; HMGS, 3-hydroxymethylglutaryl-CoA synthase; HMGR, 3-hydroxymethylglutaryl-CoA reductase 1; MK, mevalonate kinase; PMK, phospho-mevalonate kinase; MPD, mevalonate pyrophosphate decarboxylase; Idi, isopentenyl pyrophosphate isomerase. Adapted from ( ) with permission of the publisher.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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/content/10.1128/9781555816827.ch25.ch25fig02
FIGURE 2
Production of amorpha-4,11-diene via the DXP or mevalonate isoprenoid pathway and depiction of the synthetic operons used by Martin et al. ( ). The amorpha-4,11-diene pathway was separated into three plasmids, with one plasmid carrying the steps from acetyl-CoA to mevalonate, the second plasmid expressing the steps from mevalonate to FPP, and the third plasmid encoding amorpha-4,11-diene synthase (ADS). Several intermediate plasmids converting mevalonate to mevalonate-5-diphosphate (pMKPMK), to IPP (pMevB), and to DMAPP (pMBI) were constructed for comparison to pMBIS. tHMGR, truncated form of HMGR gene; A-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; MK, mevalonate kinase gene; PMK, phosphomevalonate kinase gene; MPD, mevalonate pyrophosphate decarboxylase gene; Mev-P, mevalonate phosphate; Mev-PP, mevalonate diphosphate; OPP, pyrophosphate; IPPHp, IPP, isopentenyl pyrophosphate; G3P, glyceraldehyde-3-phosphate; MEP, 2--methyl--erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2--methyl--erythritol; CDP-ME2P, 4-diphosphocytidyl-2--methyl--erythritol 2-phosphate; ME-2,4cPP, 2--methyl--erythri-tol 2,4-cyclodiphosphate; HMB4PP, 1-hydroxy-2-methyl-2-()-butenyl 4-diphosphate.
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Image of FIGURE 2
FIGURE 2

Production of amorpha-4,11-diene via the DXP or mevalonate isoprenoid pathway and depiction of the synthetic operons used by Martin et al. ( ). The amorpha-4,11-diene pathway was separated into three plasmids, with one plasmid carrying the steps from acetyl-CoA to mevalonate, the second plasmid expressing the steps from mevalonate to FPP, and the third plasmid encoding amorpha-4,11-diene synthase (ADS). Several intermediate plasmids converting mevalonate to mevalonate-5-diphosphate (pMKPMK), to IPP (pMevB), and to DMAPP (pMBI) were constructed for comparison to pMBIS. tHMGR, truncated form of HMGR gene; A-CoA, acetyl-CoA; AA-CoA, acetoacetyl-CoA; MK, mevalonate kinase gene; PMK, phosphomevalonate kinase gene; MPD, mevalonate pyrophosphate decarboxylase gene; Mev-P, mevalonate phosphate; Mev-PP, mevalonate diphosphate; OPP, pyrophosphate; IPPHp, IPP, isopentenyl pyrophosphate; G3P, glyceraldehyde-3-phosphate; MEP, 2--methyl--erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2--methyl--erythritol; CDP-ME2P, 4-diphosphocytidyl-2--methyl--erythritol 2-phosphate; ME-2,4cPP, 2--methyl--erythri-tol 2,4-cyclodiphosphate; HMB4PP, 1-hydroxy-2-methyl-2-()-butenyl 4-diphosphate.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 3
Growth curves for showing the inhibition effects caused by increasing concentrations of DL-mevalonate in Luria-Bertani medium. Strains expressing plasmids pBBR1MCS-3 (open squares), pMKPMK (circles), pMevB (diamonds), pMBI (triangles), and pMBIS (solid squares). OD600, optical density at 600 nm. Reprinted from ( ) with permission of the publisher.
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FIGURE 3

Growth curves for showing the inhibition effects caused by increasing concentrations of DL-mevalonate in Luria-Bertani medium. Strains expressing plasmids pBBR1MCS-3 (open squares), pMKPMK (circles), pMevB (diamonds), pMBI (triangles), and pMBIS (solid squares). OD600, optical density at 600 nm. Reprinted from ( ) with permission of the publisher.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 4
Growth of with increasing expression of the MevT operon. DP10 cells harbored the following plasmids (in order of increasing expression of the MevT genes): pMevT (black circles), pBAD33MevT (gray squares), and pBAD24MevT (black triangles). Cells expressing pLac33 (open circles), pBAD33 (open squares), and pBAD24 (multiplication signs) were the respective empty-plasmid controls. Reprinted from ( ) with permission of the publisher.
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FIGURE 4

Growth of with increasing expression of the MevT operon. DP10 cells harbored the following plasmids (in order of increasing expression of the MevT genes): pMevT (black circles), pBAD33MevT (gray squares), and pBAD24MevT (black triangles). Cells expressing pLac33 (open circles), pBAD33 (open squares), and pBAD24 (multiplication signs) were the respective empty-plasmid controls. Reprinted from ( ) with permission of the publisher.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 5
Mevalonate biosensor screening strategy. (a) Screening methodology. Mevalonate producers were grown in a 96-well format in C medium with inducers for 24 h. Producer cells were removed by centrifugation, and the spent medium was passed to new cultures inoculated with the biosensor. Wells containing the most mevalonate were the most fluorescent (they showed the greatest intensity of white on the plates). GFP, green fluorescent protein. (b) Mevalonate library. Seven 96-well plates are shown 15 h post-biosensor inoculation. The four white wells on each plate are mevalonate controls. Reprinted from ( ) with permission of the publisher.
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FIGURE 5

Mevalonate biosensor screening strategy. (a) Screening methodology. Mevalonate producers were grown in a 96-well format in C medium with inducers for 24 h. Producer cells were removed by centrifugation, and the spent medium was passed to new cultures inoculated with the biosensor. Wells containing the most mevalonate were the most fluorescent (they showed the greatest intensity of white on the plates). GFP, green fluorescent protein. (b) Mevalonate library. Seven 96-well plates are shown 15 h post-biosensor inoculation. The four white wells on each plate are mevalonate controls. Reprinted from ( ) with permission of the publisher.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 6
Effect of amorphadiene synthase (ADS) expression on the growth of harboring pMBIS. Cells carried pMBIS and the empty expression vector pTrc99A (without the gene) (top) or pADS expressing the amorphadiene synthase (bottom). Luria-Bertani medium was supplemented with 0, 5, 10, 20, or 40 mM DL-mevalonate. Reprinted from ( ) with permission of the publisher. OD, optical density at 600 nm.
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FIGURE 6

Effect of amorphadiene synthase (ADS) expression on the growth of harboring pMBIS. Cells carried pMBIS and the empty expression vector pTrc99A (without the gene) (top) or pADS expressing the amorphadiene synthase (bottom). Luria-Bertani medium was supplemented with 0, 5, 10, 20, or 40 mM DL-mevalonate. Reprinted from ( ) with permission of the publisher. OD, optical density at 600 nm.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 7
Flow chart of systems biology applied to achieving a production target. A host organism is selected and metabolically engineered to produce the molecule(s) of interest. The results of metabolic engineering are evaluated, for example, by monitoring phenotypes demonstrated by growth curves and product titers determined by GC. Cellular profiling is performed using measurements at all levels of the system, including mRNA, proteins, and metabolites, and flux analysis to identify bottlenecks in the pathway. Once bottlenecks are identified, another round of engineering is carried out to overcome the limitations. The cycle is completed once the desired production level has been achieved. OD, optical density at 600 nm. Reprinted from ( ) with permission of the publisher.
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FIGURE 7

Flow chart of systems biology applied to achieving a production target. A host organism is selected and metabolically engineered to produce the molecule(s) of interest. The results of metabolic engineering are evaluated, for example, by monitoring phenotypes demonstrated by growth curves and product titers determined by GC. Cellular profiling is performed using measurements at all levels of the system, including mRNA, proteins, and metabolites, and flux analysis to identify bottlenecks in the pathway. Once bottlenecks are identified, another round of engineering is carried out to overcome the limitations. The cycle is completed once the desired production level has been achieved. OD, optical density at 600 nm. Reprinted from ( ) with permission of the publisher.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 8
Transcript profiles of the initial steps of type II FAB in . Malonyl-CoA is synthesized from acetyl-CoA by the action of acetyl-CoA carboxylase, a heterotetramer composed of subunits encoded by . The malonate moiety is transferred from CoA to the acyl carrier protein (ACP) by the action of malonyl-CoA:ACP transacylase (FabD). Also shown (inset) are the expression values and scores (in parentheses) for FAB genes that exhibited biologically significant upregulation in the mevalonate-producing strain ( DP10 containing pBAD33MevT and pBAD18) relative to the inactive-pathway control strain [ DP10 containing pMevT(C159A) and pBAD18] in the microarray analysis. Values for the control strain were set at 1.0. Adapted from ( ).
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FIGURE 8

Transcript profiles of the initial steps of type II FAB in . Malonyl-CoA is synthesized from acetyl-CoA by the action of acetyl-CoA carboxylase, a heterotetramer composed of subunits encoded by . The malonate moiety is transferred from CoA to the acyl carrier protein (ACP) by the action of malonyl-CoA:ACP transacylase (FabD). Also shown (inset) are the expression values and scores (in parentheses) for FAB genes that exhibited biologically significant upregulation in the mevalonate-producing strain ( DP10 containing pBAD33MevT and pBAD18) relative to the inactive-pathway control strain [ DP10 containing pMevT(C159A) and pBAD18] in the microarray analysis. Values for the control strain were set at 1.0. Adapted from ( ).

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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FIGURE 9
Flow chart of metabolic engineering efforts for the high-level production of precursors to artemisinin.
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Image of FIGURE 9
FIGURE 9

Flow chart of metabolic engineering efforts for the high-level production of precursors to artemisinin.

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
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Tables

Metabolic Engineering of Escherichia coli for the Production of a Precursor to Artemisinin, an Antimalarial Drug
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Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25
/content/10.1128/9781555816827.ch25.ch25tab01
TABLE 1
Plasmids used for metabolic engineering of for production of precursors to artemisinin
Generic image for table
TABLE 1

Plasmids used for metabolic engineering of for production of precursors to artemisinin

Citation: Petzold C, Keasling J. 2010. Metabolic Engineering of for the Production of a Precursor to Artemisinin, an Antimalarial Drug, p 364-379. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch25

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