Journal
BIORESOURCE TECHNOLOGY
Volume 245, Issue -, Pages 1327-1333Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.biortech.2017.05.004
Keywords
AMA1; GoldenMOCS; Aconitic acid; Modular cloning; CRISPR/Cas9
Funding
- Federal Ministry of Science, Research and Economy (BMWFW)
- Federal Ministry of Traffic, Innovation and Technology (bmvit)
- Styrian Business Promotion Agency SFG
- Standortagentur Tirol
- Government of Lower Austria
- Business Agency Vienna through the COMET
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Metabolic engineering requires functional genetic tools for easy and quick generation of multiple pathway variants. A genetic engineering toolbox for A. niger is presented, which facilitates the generation of strains carrying heterologous expression cassettes at a defined genetic locus. The system is compatible with Golden Gate cloning, which facilitates the DNA construction process and provides high design flexibility. The integration process is mediated by a CRISPR/Cas9 strategy involving the cutting of both the genetic integration locus (pyrG) as well as the integrating plasmid. Only a transient expression of Cas9 is necessary and the carrying plasmid is readily lost using a size-reduced AMA1 variant. A high integration efficiency into the fungal genome of up to 100% can be achieved, thus reducing the screening process significantly. The feasibility of the approach was demonstrated by the integration of an expression cassette enabling the production of aconitic acid in A. niger. (C) 2017 Elsevier Ltd. All rights reserved.
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