<div class="csl-bib-body">
<div class="csl-entry">Sitara, A., Hocq, R. V., & Pflügl, S. (2024, September 25). <i>Exploiting CRISPR in Thermoanaerobacter kivui</i> [Conference Presentation]. 14th International Congress Extremophiles 2024, Loutraki, Greece.</div>
</div>
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/204258
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dc.description.abstract
Acetogens are autotrophic microbes, attractive for industrial, carbon-negative production of value-added chemicals. Thermoanaerobacter kivui is a fast-growing, thermophilic acetogen, able to utilize gaseous C1 compounds, such as CO2, CO (after adaptation), and H2, which are available in large quantities as industrial steel mill off-gasses or can be obtained via gasification from biogenic residues. Combined with its genetic tractability, T. kivui is an ideal host for metabolic engineering towards industrial production of chemicals of interest.
Current genome editing methods rely on homologous recombination with auxotrophy-based selection, which requires multiple subculturing steps and can be labor-intensive. To fully exploit T. kivui potential for industrial biotechnology, developing more efficient genome editing tools would be desirable.
In this work, we created a genome editing method based on the endogenous CRISPR Type I-B system of T. kivui. With this method, a deletion (pyrE used as the proof-of-concept) or insertion (pFAST) is possible in a single plating step with a transformation efficiency of 106 CFU/μg/ml and 100% editing efficiency. With this tool, the speed of genome editing in T. kivui was drastically accelerated, with a current average speed of 1 genome-edited strain per month, which is also increasing. These results render T. kivui endogenous CRISPR-based genome editing the most efficient among the thermophilic anaerobes. Additionally, conventional hosts used for plasmid cloning, such as E. coli, can be bypassed without loss in efficiency, as the assembled DNA fragments can be transformed directly to T. kivui.
The redox-sensing factor rex and the heat-responsive transcriptional repressor hrcA were targeted with CRISPR for deletion, and phenotypic analysis of the resulting mutants was performed to illuminate the role of these transcription factors in T. kivui. Overall, the developed highly efficient CRISPR system should facilitate iterative genetic engineering approaches in the future and could potentially be applied to other thermophilic hosts.
en
dc.description.sponsorship
FWF - Österr. Wissenschaftsfonds
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dc.language.iso
en
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dc.subject
CRISPR
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dc.subject
Acetogens
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dc.subject
genome editing
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dc.subject
Thermophile
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dc.subject
Thermoanaerobacter kivui
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dc.title
Exploiting CRISPR in Thermoanaerobacter kivui
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dc.type
Presentation
en
dc.type
Vortrag
de
dc.relation.grantno
Y1513-N
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dc.type.category
Conference Presentation
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tuw.project.title
Formiat-basierte acetogene Bioproduktion von Treibstoffen und Chemikalien
