ArticlePloS one2013
Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA) method.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed.
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- Biosensor-driven, model-based optimization of the orthogonally expressed naringenin biosynthesis pathway.Microbial cell factories · 2022Article
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- A highly efficient non-viral process for programming mesenchymal stem cells for gene directed enzyme prodrug cancer therapy.Scientific reports · 2020Article
- Emerging molecular biology tools and strategies for engineering natural product biosynthesis.Metabolic engineering communications · 2020Review
- Modular engineering for microbial production of carotenoids.Metabolic engineering communications · 2020Review
- Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis.Microbial cell factories · 2019Article
- A standard for near-scarless plasmid construction using reusable DNA parts.Nature communications · 2019Article
- Multidimensional heuristic process for high-yield production of astaxanthin and fragrance molecules in Escherichia coli.Nature communications · 2018Article
- A sigma factor toolbox for orthogonal gene expression in Escherichia coli.Nucleic acids research · 2018Article
- Multienzyme Biosynthesis of Dihydroartemisinic Acid.Molecules (Basel, Switzerland) · 2017Article
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- Molecular Cloning Designer Simulator (MCDS): All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects.Metabolic engineering communications · 2016Article
- Systems analysis of methylerythritol-phosphate pathway flux in E. coli: insights into the role of oxidative stress and the validity of lycopene as an isoprenoid reporter metabolite.Microbial cell factories · 2015Article
- Metabolic engineering of Escherichia coli into a versatile glycosylation platform: production of bio-active quercetin glycosides.Microbial cell factories · 2015Article
- Combinatorial optimization of synthetic operons for the microbial production of p-coumaryl alcohol with Escherichia coli.Microbial cell factories · 2015Article
- Distributing a metabolic pathway among a microbial consortium enhances production of natural products.Nature biotechnology · 2015Article
- A novel approach for metabolic pathway optimization: Oligo-linker mediated assembly (OLMA) method.Journal of biological engineering · 2015Article
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4 authors.
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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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