Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

Chung, Daehwan; Farkas, Joel; Huddleston, Jennifer R.; Olivar, Estefania; Westpheling, Janet

doi:10.1371/journal.pone.0043844

Cited by 74 publications

(122 citation statements)

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“…this strain no longer produced lactate, instead diverting metabolic flux to additional acetate and H 2 , demonstrating the utility of the newly developed tools to provide a platform for further strain engineering. The recent development of genetic methods for the manipulation of this organism (19,21,22) opens the door for metabolic engineering for the direct conversion of unpretreated plant biomass to liquid fuels, such as ethanol, via "single step bioprocessing" (Fig. 1).…”

Section: Significancementioning

confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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198

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Ethanol is the most widely used renewable transportation biofuel in the United States, with the production of 13.3 billion gallons in 2012 [John UM (2013) Contribution of the Ethanol Industry to the Economy of the United States]. Despite considerable effort to produce fuels from lignocellulosic biomass, chemical pretreatment and the addition of saccharolytic enzymes before microbial bioconversion remain economic barriers to industrial deployment [Lynd LR, et al. (2008) Nat Biotechnol 26(2):169-172]. We began with the thermophilic, anaerobic, cellulolytic bacterium Caldicellulosiruptor bescii, which efficiently uses unpretreated biomass, and engineered it to produce ethanol. Here we report the direct conversion of switchgrass, a nonfood, renewable feedstock, to ethanol without conventional pretreatment of the biomass. This process was accomplished by deletion of lactate dehydrogenase and heterologous expression of a Clostridium thermocellum bifunctional acetaldehyde/alcohol dehydrogenase. Whereas wild-type C. bescii lacks the ability to make ethanol, 70% of the fermentation products in the engineered strain were ethanol [12.8 mM ethanol directly from 2% (wt/vol) switchgrass, a real-world substrate] with decreased production of acetate by 38% compared with wild-type. Direct conversion of biomass to ethanol represents a new paradigm for consolidated bioprocessing, offering the potential for carbon neutral, cost-effective, sustainable fuel production. metabolic engineering | bioenergy and thermophiles

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Section: Significancementioning

confidence: 99%

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Chung

Cha

Guss

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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198

171

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“…Its ability to deconstruct lignocellulosic biomass, combined with its high optimal growth temperature, makes C. bescii of great biotechnological interest for metabolic engineering efforts toward lignocellulosic bio-based fuel and chemical production. To this end, a genetic system was developed for C. bescii utilizing a uracil auxotrophic mutant background strain and the counterselectable marker pyrF, a gene required for biosynthesis of uracil that also confers sensitivity to 5-fluoroorotic acid (5-FOA) (2,3). Because there was no method for direct selection of a targeted deletion of pyrF in wild-type C. bescii, the initial development of a genetic background strain relied on the selection of random mutants containing deletions in uracil biosynthesis pathway genes (4).…”

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confidence: 99%

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

Williams-Rhaesa

Poole

Dinsmore

et al. 2017

Appl Environ Microbiol

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Caldicellulosiruptor bescii is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA, and the second (MACB1018) is based on the targeted deletion of pyrE, making use of a kanamycin resistance marker. Importantly, an active insertion element, ISCbe4, was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4 movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of C. bescii from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in C. bescii. Furthermore, a facile approach for assessing genomic stability in C. bescii has been established.IMPORTANCE Caldicellulosiruptor bescii is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in C. bescii has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes (pyrFA) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these ISCbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies.

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“…It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production (3,4). Such organisms require a tractable genetic system for metabolic engineering applications, and such a system has been established for C. bescii in recent years (5,6). A major step in developing a genetic system for C. bescii was the discovery that it has a restriction-modification system that severely limits its transformability (7).…”

mentioning

confidence: 99%

“…A major step in developing a genetic system for C. bescii was the discovery that it has a restriction-modification system that severely limits its transformability (7). This barrier to transformation was overcome through the use of a methylated donor plasmid with a methylation pattern that protected the transformed DNA from restriction digestion (5). The system was further improved by deleting the gene encoding the restriction enzyme, CbeI, making it possible to use nonmethylated donor DNA in transformation (6).…”

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confidence: 99%

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A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

Lipscomb

Conway

Blumer-Schuette

et al. 2016

Appl Environ Microbiol

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Caldicellulosiruptor bescii, an anaerobic Gram-positive bacterium with an optimal growth temperature of 78°C, is the most thermophilic cellulose degrader known. It is of great biotechnological interest, as it efficiently deconstructs nonpretreated lignocellulosic plant biomass. Currently, its genetic manipulation relies on a mutant uracil auxotrophic background strain that contains a random deletion in the pyrF genome region. The pyrF gene serves as a genetic marker to select for uracil prototrophy, and it can also be counterselected for loss via resistance to the compound 5-fluoroorotic acid (5-FOA). To expand the C. bescii genetic tool kit, kanamycin resistance was developed as a selection for genetic manipulation. A codon-optimized version of the highly thermostable kanamycin resistance gene (named Cbhtk) allowed the use of kanamycin selection to obtain transformants of either replicating or integrating vector constructs in C. bescii. These strains showed resistance to kanamycin at concentrations >50 g · ml ؊1 , whereas wild-type C. bescii was sensitive to kanamycin at 10 g · ml ؊1 . In addition, placement of the Cbhtk marker between homologous recombination regions in an integrating vector allowed direct selection of a chromosomal mutation using both kanamycin and 5-FOA. Furthermore, the use of kanamycin selection enabled the targeted deletion of the pyrE gene in wild-type C. bescii, generating a uracil auxotrophic genetic background strain resistant to 5-FOA. The pyrE gene functioned as a counterselectable marker, like pyrF, and was used together with Cbhtk in the ⌬pyrE background strain to delete genes encoding lactate dehydrogenase and the CbeI restriction enzyme. IMPORTANCECaldicellulosiruptor bescii is a thermophilic anaerobic bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass. It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel production. The current genetic system used with C. bescii is based upon only a single selection strategy, and this uses the gene involved in a primary biosynthetic pathway. There are many advantages with an additional genetic selection using an antibiotic. This presents a challenge for thermophilic microorganisms, as only a limited number of antibiotics are stable above 50°C, and a thermostable version of the enzyme conferring antibiotic resistance must be obtained. In this work, we have developed a selection system for C. bescii using the antibiotic kanamycin and have shown that, in combination with the biosynthetic gene marker, it can be used to efficiently delete genes in this organism. Caldicellulosiruptor bescii is a thermophilic anaerobic Grampositive bacterium with an optimal growth temperature of 78°C, and it has the ability to efficiently deconstruct nonpretreated lignocellulosic plant biomass (1, 2). It is, therefore, of biotechnological interest for genetic engineering applications geared toward biofuel product...

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Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

Cited by 74 publications

References 64 publications

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

Genome Stability in Engineered Strains of the Extremely Thermophilic Lignocellulose-Degrading Bacterium Caldicellulosiruptor bescii

A Highly Thermostable Kanamycin Resistance Marker Expands the Tool Kit for Genetic Manipulation of Caldicellulosiruptor bescii

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