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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 cross-lapping in vitro assembly (CLIVA) method.(A) Illustration of the design at one junction between two modules (blue and red). The cross-lapping primer consists of gene specific sequence (GSS) and tag sequence complementary to adjacent primer’s GSS. The phosphorothioate modifications were indicated as cycles. An “Ox/y” designation was used to define the primers, where O denoted overlap; x was the length of overlap which had one modification at each y base pairs of the sequence. (B) Illustration of assembling of multiple DNA modules into one plasmid.
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pone-0079557-g002: The cross-lapping in vitro assembly (CLIVA) method.(A) Illustration of the design at one junction between two modules (blue and red). The cross-lapping primer consists of gene specific sequence (GSS) and tag sequence complementary to adjacent primer’s GSS. The phosphorothioate modifications were indicated as cycles. An “Ox/y” designation was used to define the primers, where O denoted overlap; x was the length of overlap which had one modification at each y base pairs of the sequence. (B) Illustration of assembling of multiple DNA modules into one plasmid.

Mentions: PCR has been used to produce overlapping homologous sequences by adding extraneous tag sequences to the gene specific primers [3,12,15]. With such a design, the homologous sequences are limited to the length of the tags. In order to increase the assembly efficiency, we designed the tags to be homologous to the gene specific sequences (Figure 1A). This cross-lapping design allowed us to increase the length of the homologous sequences at each junction as compared to conventional strategies. Besides, other than modifying all the bases in the homologous sequences which increased the cost of primer synthesis, we explored the possibility of decreasing the modification frequency (number of phosphothiodate modification per oligonucleotide) while maintaining a high efficiency of assembly (Figure 2A). Critically, by the use of certain cations, the efficiency of the assembly process was substantially increased and this has enabled the construction of large plasmids from multiple fragments in one step.


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 cross-lapping in vitro assembly (CLIVA) method.(A) Illustration of the design at one junction between two modules (blue and red). The cross-lapping primer consists of gene specific sequence (GSS) and tag sequence complementary to adjacent primer’s GSS. The phosphorothioate modifications were indicated as cycles. An “Ox/y” designation was used to define the primers, where O denoted overlap; x was the length of overlap which had one modification at each y base pairs of the sequence. (B) Illustration of assembling of multiple DNA modules into one plasmid.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079557-g002: The cross-lapping in vitro assembly (CLIVA) method.(A) Illustration of the design at one junction between two modules (blue and red). The cross-lapping primer consists of gene specific sequence (GSS) and tag sequence complementary to adjacent primer’s GSS. The phosphorothioate modifications were indicated as cycles. An “Ox/y” designation was used to define the primers, where O denoted overlap; x was the length of overlap which had one modification at each y base pairs of the sequence. (B) Illustration of assembling of multiple DNA modules into one plasmid.
Mentions: PCR has been used to produce overlapping homologous sequences by adding extraneous tag sequences to the gene specific primers [3,12,15]. With such a design, the homologous sequences are limited to the length of the tags. In order to increase the assembly efficiency, we designed the tags to be homologous to the gene specific sequences (Figure 1A). This cross-lapping design allowed us to increase the length of the homologous sequences at each junction as compared to conventional strategies. Besides, other than modifying all the bases in the homologous sequences which increased the cost of primer synthesis, we explored the possibility of decreasing the modification frequency (number of phosphothiodate modification per oligonucleotide) while maintaining a high efficiency of assembly (Figure 2A). Critically, by the use of certain cations, the efficiency of the assembly process was substantially increased and this has enabled the construction of large plasmids from multiple fragments in one step.

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