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A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae

Paper ID Volume ID Publish Year Pages File Format Full-Text
31473 44800 2016 9 PDF Available
Title
A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae
Abstract

•Created a novel approach for integration of DNA at delta sites of yeast genome.•Enabled efficient multi-copy, one-step, markerless integration of large DNA.•Achieved an unprecedented 18-copy genomic integration of a 24 kb pathway in one step.•The platform surpassed current strategies and will be a useful genome editing tool.

Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.

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Keywords
CRISPR-Cas; Delta integration; Multi-copy integration; Genome editing; Saccharomyces cerevisiae
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A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 33, January 2016, Pages 19–27
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
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us