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Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA) method.

Zou R, Zhou K, Stephanopoulos G, Too HP - PLoS ONE (2013)

Bottom Line: Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene.Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway.The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene.

View Article: PubMed Central - PubMed

Affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, Singapore, Singapore.

ABSTRACT
The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10(3) cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.

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Assembly of DXP pathway.(A) The dxp pathway and Fe-S cluster assembling pathway. GA3P: glyceraldehyde 3-phosphate, DXP: 1-deoxy-D-xylulose 5-phosphate, MEP: 2C-methyl-D-erythritol 4-phosphate, CDP-ME: 4-diphosphocytidyl-2C-methyl D-erythritol, CDP-MEP: 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, MEC: 2C-methyl-D-erythritol 2,4-diphosphate, HMBPP: hydroxylmethylbutenyl diphosphate, IPP: Isopentenyl pyrophosphate, DMAPP: Dimethylallyl pyrophosphate, GPP: Geranyl diphosphate, FPP: Farnesyl diphosphate, GGPP: Geranylgeranyl diphosphate. (B) Illustration of various modules assembled in the project (correlated to Table S3). CAM: chloramphenicol resistance gene, p15A-ori: p15A original of replication.
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pone-0079557-g001: Assembly of DXP pathway.(A) The dxp pathway and Fe-S cluster assembling pathway. GA3P: glyceraldehyde 3-phosphate, DXP: 1-deoxy-D-xylulose 5-phosphate, MEP: 2C-methyl-D-erythritol 4-phosphate, CDP-ME: 4-diphosphocytidyl-2C-methyl D-erythritol, CDP-MEP: 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, MEC: 2C-methyl-D-erythritol 2,4-diphosphate, HMBPP: hydroxylmethylbutenyl diphosphate, IPP: Isopentenyl pyrophosphate, DMAPP: Dimethylallyl pyrophosphate, GPP: Geranyl diphosphate, FPP: Farnesyl diphosphate, GGPP: Geranylgeranyl diphosphate. (B) Illustration of various modules assembled in the project (correlated to Table S3). CAM: chloramphenicol resistance gene, p15A-ori: p15A original of replication.

Mentions: In this study, the effects of various combinations of the enzymes in the DXP pathway in providing precursors to downstream production of amorphadiene, the precursor for antimalarial drug artemisinin [30], was systematically investigated for the first time (Figure 1A). The CLIVA method enabled the assembly of multiple plasmids containing various combinations of genes rapidly. Metabolic profiling using ultra-performance liquid chromatography mass spectrometry (UPLC-MS) [31] identified the accumulation of intracellular MEC (one of the DXP pathway intermediate) as a limiting factor for isoprenoid production. The overexpression of iron sulfur cluster (Isc) operon, which supplied the cofactors for the function of two succeeding enzymes downstream of MEC (ispG and ispH) (Figure 1A), was found to modestly enhance the production of amorphadiene.


Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA) method.

Zou R, Zhou K, Stephanopoulos G, Too HP - PLoS ONE (2013)

Assembly of DXP pathway.(A) The dxp pathway and Fe-S cluster assembling pathway. GA3P: glyceraldehyde 3-phosphate, DXP: 1-deoxy-D-xylulose 5-phosphate, MEP: 2C-methyl-D-erythritol 4-phosphate, CDP-ME: 4-diphosphocytidyl-2C-methyl D-erythritol, CDP-MEP: 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, MEC: 2C-methyl-D-erythritol 2,4-diphosphate, HMBPP: hydroxylmethylbutenyl diphosphate, IPP: Isopentenyl pyrophosphate, DMAPP: Dimethylallyl pyrophosphate, GPP: Geranyl diphosphate, FPP: Farnesyl diphosphate, GGPP: Geranylgeranyl diphosphate. (B) Illustration of various modules assembled in the project (correlated to Table S3). CAM: chloramphenicol resistance gene, p15A-ori: p15A original of replication.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818232&req=5

pone-0079557-g001: Assembly of DXP pathway.(A) The dxp pathway and Fe-S cluster assembling pathway. GA3P: glyceraldehyde 3-phosphate, DXP: 1-deoxy-D-xylulose 5-phosphate, MEP: 2C-methyl-D-erythritol 4-phosphate, CDP-ME: 4-diphosphocytidyl-2C-methyl D-erythritol, CDP-MEP: 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate, MEC: 2C-methyl-D-erythritol 2,4-diphosphate, HMBPP: hydroxylmethylbutenyl diphosphate, IPP: Isopentenyl pyrophosphate, DMAPP: Dimethylallyl pyrophosphate, GPP: Geranyl diphosphate, FPP: Farnesyl diphosphate, GGPP: Geranylgeranyl diphosphate. (B) Illustration of various modules assembled in the project (correlated to Table S3). CAM: chloramphenicol resistance gene, p15A-ori: p15A original of replication.
Mentions: In this study, the effects of various combinations of the enzymes in the DXP pathway in providing precursors to downstream production of amorphadiene, the precursor for antimalarial drug artemisinin [30], was systematically investigated for the first time (Figure 1A). The CLIVA method enabled the assembly of multiple plasmids containing various combinations of genes rapidly. Metabolic profiling using ultra-performance liquid chromatography mass spectrometry (UPLC-MS) [31] identified the accumulation of intracellular MEC (one of the DXP pathway intermediate) as a limiting factor for isoprenoid production. The overexpression of iron sulfur cluster (Isc) operon, which supplied the cofactors for the function of two succeeding enzymes downstream of MEC (ispG and ispH) (Figure 1A), was found to modestly enhance the production of amorphadiene.

Bottom Line: Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene.Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway.The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene.

View Article: PubMed Central - PubMed

Affiliation: Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, Singapore, Singapore.

ABSTRACT
The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10(3) cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.

Show MeSH