ArticleBiotechnology for biofuels2014
Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme.
Article in Biotechnology for biofuels, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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14 citing papers in PubMed, 29 citations in OpenAlex.
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- Efficient Secretion of Murine IL-2 From an Attenuated Strain ofFrontiers in microbiology · 2021Article
- CRISPR interference (CRISPRi) as transcriptional repression tool for Hungateiclostridium thermocellum DSM 1313.Microbial biotechnology · 2020Article
- Improved electrocompetence and metabolic engineering ofEngineering in life sciences · 2019Article
- Recent Developments of the Synthetic Biology Toolkit forFrontiers in microbiology · 2018Review
- Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.Applied and environmental microbiology · 2017Article
- Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium.Applied and environmental microbiology · 2016Article
- A roadmap for gene system development in Clostridium.Anaerobe · 2016Review
- Genome-directed analysis of prophage excision, host defence systems, and central fermentative metabolism in Clostridium pasteurianum.Scientific reports · 2016Article
- Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum.Applied and environmental microbiology · 2016Article
- Consolidating biofuel platforms through the fermentative bioconversion of crude glycerol to butanol.World journal of microbiology & biotechnology · 2016Review
- Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium.Scientific reports · 2016Article
- Using Group II Introns for Attenuating the In Vitro and In Vivo Expression of a Homing Endonuclease.PloS one · 2016Article
- Whole-genome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth.Biotechnology for biofuels · 2015Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundClostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum.
resultsHere we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism's methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5'-AAGNNNNNCTCC-3' (N = A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5'-CGCG-3'). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting ΔcpaAIR mutant strain was efficiently electrotransformed with M.FnuDII-unmethylated plasmid DNA.
conclusionsThe markerless and plasmidless ΔcpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum.
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