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One step DNA assembly for combinatorial metabolic engineering

Paper ID Volume ID Publish Year Pages File Format Full-Text
31538 44810 2014 8 PDF Available
Title
One step DNA assembly for combinatorial metabolic engineering
Abstract

•We developed a single strand assembly (SSA) method for combinatorial pathway engineering.•The SSA method was applied to create promoter, RBS and/or mutant enzyme libraries.•This SSA method was applied to fine-tune multi-gene pathways generating an orthogonal gene expression.•We demonstrated that SSA is a rapid, reliable, easy-to-use and amendable method for standardization and automation.•State-of-the-art promoter, RBS and promoter-RBS library approaches and outcomes were compared.

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity.

Keywords
Metabolic engineering; Pathway optimization; Promoter library; RBS library; Protein library
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One step DNA assembly for combinatorial metabolic engineering
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 23, May 2014, Pages 70–77
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
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Full-text PDF Download
Online Support
Any Questions? feel free to contact us