A novel SfaNI-like restriction-modification system in Caldicellulosiruptor extents the genetic engineering toolbox for this genus.

Autor: Swinnen S; BluCon Biotech GmbH, Cologne, Germany., Zurek C; BRAIN Biotech AG, Zwingenberg, Germany., Krämer M; BluCon Biotech GmbH, Cologne, Germany., Heger RM; BRAIN Biotech AG, Zwingenberg, Germany., Domeyer JE; BRAIN Biotech AG, Zwingenberg, Germany., Ziegler J; BRAIN Biotech AG, Zwingenberg, Germany., Svetlitchnyi VA; BluCon Biotech GmbH, Cologne, Germany., Läufer A; BluCon Biotech GmbH, Cologne, Germany.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2022 Dec 29; Vol. 17 (12), pp. e0279562. Date of Electronic Publication: 2022 Dec 29 (Print Publication: 2022).
DOI: 10.1371/journal.pone.0279562
Abstrakt: Caldicellulosiruptor is a genus of thermophilic to hyper-thermophilic microorganisms that express and secrete an arsenal of enzymes degrading lignocellulosic biomasses into fermentable sugars. Because of this distinguished feature, strains of Caldicellulosiruptor have been considered as promising candidates for consolidated bioprocessing. Although a few Caldicellulosiruptor strains with industrially relevant characteristics have been isolated to date, it is apparent that further improvement of the strains is essential for industrial application. The earlier identification of the HaeIII-like restriction-modification system in C. bescii strain DSM 6725 has formed the basis for genetic methods with the aim to improve the strain's lignocellulolytic activity and ethanol production. In this study, a novel SfaNI-like restriction-modification system was identified in Caldicellulosiruptor sp. strain BluCon085, consisting of an endonuclease and two methyltransferases that recognize the reverse-complement sequences 5'-GATGC-3' and 5'-GCATC-3'. Methylation of the adenine in both sequences leads to an asymmetric methylation pattern in the genomic DNA of strain BluCon085. Proteins with high percentage of identity to the endonuclease and two methyltransferases were identified in the genomes of C. saccharolyticus strain DSM 8903, C. naganoensis strain DSM 8991, C. changbaiensis strain DSM 26941 and Caldicellulosiruptor sp. strain F32, suggesting that a similar restriction-modification system may be active also in these strains and respective species. We show that methylation of plasmid and linear DNA by the identified methyltransferases, obtained by heterologous expression in Escherichia coli, is sufficient for successful transformation of Caldicellulosiruptor sp. strain DIB 104C. The genetic engineering toolbox developed in this study forms the basis for rational strain improvement of strain BluCon085, a derivative from strain DIB 104C with exceptionally high L-lactic acid production. The toolbox may also work for other species of the genus Caldicellulosiruptor that have so far not been genetically tractable.
Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: All authors were employees of BluCon Biotech GmbH or BRAIN Biotech AG at the moment that the study was conceived and carried out.
(Copyright: © 2022 Swinnen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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