scholarly journals Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum

2021 ◽  
Vol 22 (7) ◽  
pp. 3704
Author(s):  
Tom Wilding-Steele ◽  
Quentin Ramette ◽  
Paul Jacottin ◽  
Philippe Soucaille

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

Gene ◽  
1995 ◽  
Vol 155 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Christopher K. Murphy ◽  
Eric J. Stewart ◽  
Jon Beckwith

2012 ◽  
Vol 17 (4) ◽  
pp. 535-541 ◽  
Author(s):  
Gregory J. Crowther ◽  
S. Arshiya Quadri ◽  
Benjamin J. Shannon-Alferes ◽  
Wesley C. Van Voorhis ◽  
Henry Rosen

More than 20% of bacterial proteins are noncytoplasmic, and most of these pass through the SecYEG channel en route to the periplasm, cell membrane, or surrounding environment. The Sec pathway, encompassing SecYEG and several associated proteins (SecA, SecB, YidC, SecDFYajC), is of interest as a potential drug target because it is distinct from targets of current drugs, is essential for bacterial growth, and exhibits dissimilarities in eukaryotes and bacteria that increase the likelihood of selectively inhibiting the microbial pathway. As a step toward validating the pathway as a drug target, we have adapted a mechanism-based whole-cell assay in a manner suitable for high-throughput screening (HTS). The assay uses an engineered strain of Escherichia coli that accumulates beta-galactosidase (β-gal) in its cytoplasm if translocation through SecYEG is blocked. The assay should facilitate rapid identification of compounds that specifically block the Sec pathway because widely, toxic compounds and nonspecific protein synthesis inhibitors prevent β-gal production and thus do not register as hits. Testing of current antibiotics confirmed that they do not generally act through the Sec pathway. A mini-screen of 800 compounds indicated the assay’s readiness for larger screening projects.


2008 ◽  
Vol 40 (2) ◽  
pp. 312-320 ◽  
Author(s):  
Soo Yun Moon ◽  
Soon Ho Hong ◽  
Tae Yong Kim ◽  
Sang Yup Lee

2017 ◽  
Vol 241 ◽  
pp. 430-438 ◽  
Author(s):  
Chonglong Wang ◽  
Bakht Zada ◽  
Gongyuan Wei ◽  
Seon-Won Kim

2007 ◽  
Vol 76 (5) ◽  
pp. 1051-1057 ◽  
Author(s):  
Cassandra De Muynck ◽  
Jef Van der Borght ◽  
Marjan De Mey ◽  
Sofie L. De Maeseneire ◽  
Inge N. A. Van Bogaert ◽  
...  

2011 ◽  
Vol 40 (8) ◽  
pp. 3524-3537 ◽  
Author(s):  
Ana I. Prieto ◽  
Christina Kahramanoglou ◽  
Ruhi M. Ali ◽  
Gillian M. Fraser ◽  
Aswin S. N. Seshasayee ◽  
...  

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