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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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The performance of different combinations of DXP pathway genes in E. coli.(A) 48h amorphadiene yield. Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors were shown. (B) The correlation of pathway modules with amorphadiene yield at optimal IPTG inductions. (C) Early response of intracellular metabolites at 3h after induction. The gray areas indicated the overexpressed section of DXP pathway. The experiment was repeated twice and the averages were shown.
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pone-0079557-g004: The performance of different combinations of DXP pathway genes in E. coli.(A) 48h amorphadiene yield. Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors were shown. (B) The correlation of pathway modules with amorphadiene yield at optimal IPTG inductions. (C) Early response of intracellular metabolites at 3h after induction. The gray areas indicated the overexpressed section of DXP pathway. The experiment was repeated twice and the averages were shown.

Mentions: Next, the various combinations of pathway genes with the essential module (IAA) containing the heterologous amorphadiene synthase were tested for amorphadiene production. Consistent with previous reports [31,39,40], high induction resulted in lower production of isoprenoids (Figure 4A, different IPTG inductions). Comparing constructs at their optimal induction levels, as expected, the expression of the first committed step (dxs – module S) enhanced the amorphadiene production. However, the overexpression of the rest of the pathway genes in conjunction with the S and IAA modules had variable negative effects on productivity. Notably, the expression of GH module (ispG and ispH) as well as R (dxr) -DEF (ispD, ispE and ispF) modules led to a significant inhibition on the production (Figure 4A). Consistent with the observations, a simple linear model correlating the pathway modules and amorphadiene yields at their optimal inductions revealed that the expression of GH module or the co-expression of R-DEF modules had negative impacts (Figure 4B).


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)

The performance of different combinations of DXP pathway genes in E. coli.(A) 48h amorphadiene yield. Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors were shown. (B) The correlation of pathway modules with amorphadiene yield at optimal IPTG inductions. (C) Early response of intracellular metabolites at 3h after induction. The gray areas indicated the overexpressed section of DXP pathway. The experiment was repeated twice and the averages were shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079557-g004: The performance of different combinations of DXP pathway genes in E. coli.(A) 48h amorphadiene yield. Different concentrations of IPTG were represented by bars with different colors. The experiment was repeated four times and the standard errors were shown. (B) The correlation of pathway modules with amorphadiene yield at optimal IPTG inductions. (C) Early response of intracellular metabolites at 3h after induction. The gray areas indicated the overexpressed section of DXP pathway. The experiment was repeated twice and the averages were shown.
Mentions: Next, the various combinations of pathway genes with the essential module (IAA) containing the heterologous amorphadiene synthase were tested for amorphadiene production. Consistent with previous reports [31,39,40], high induction resulted in lower production of isoprenoids (Figure 4A, different IPTG inductions). Comparing constructs at their optimal induction levels, as expected, the expression of the first committed step (dxs – module S) enhanced the amorphadiene production. However, the overexpression of the rest of the pathway genes in conjunction with the S and IAA modules had variable negative effects on productivity. Notably, the expression of GH module (ispG and ispH) as well as R (dxr) -DEF (ispD, ispE and ispF) modules led to a significant inhibition on the production (Figure 4A). Consistent with the observations, a simple linear model correlating the pathway modules and amorphadiene yields at their optimal inductions revealed that the expression of GH module or the co-expression of R-DEF modules had negative impacts (Figure 4B).

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