【摘 要】
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Imbalances in gene expression of a metabolic pathway can result in less-yield of the desired products.Several targets were intensively investigated to balan
【机 构】
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ChineseAcademyofSciencesKeyLaboratoryofMicrobialPhysiologicalandMetabolicEngineering,InstituteofMicr
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Imbalances in gene expression of a metabolic pathway can result in less-yield of the desired products.Several targets were intensively investigated to balance the gene expression, such as promoter, ribosome binding site (RBS), the order of genes, as well as the species of the enzymes.Here, we reported a new DNA assembling method to vary all the regulatory targets simultaneously, named oligo-linker mediated assembly (OLMA) method.A unique feature of the method is the design of a chemically synthetic double-stranded DNA library to achieve the variations of promoter and RBS,and to facilitate the swapping of the gene order, resulting in a PCR-free and barcode-free DNA assembly approach.Two experimental cases were used to demonstrate the capability of the method: (1) assembly of multiple pieces of LacZ expression cassette;(2) optimization of four enzymes in lycopene biosynthetic pathway.Our results indicated that the OLMA method not only exploited larger combinatorial space, but also reduced the inefficient mutants.
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