Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

c Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO 2 , and H 2 . Caldicellulosiruptor bescii, C. kronotskyensis, and C. saccharolyticus solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (Panicum virgatum) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with C. saccharolyticus, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation. T he genusCaldicellulosiruptor is comprised of Gram-positive, anaerobic bacteria that ferment a variety of simple and complex carbohydrates to primarily H 2 , CO 2 , acetate, and lactate at temperatures at or above 70°C (1). To date, Caldicellulosiruptor species have been isolated globally from terrestrial hot springs and thermal features in locations including the United States (C. owensensis [2,3] and C. obsidiansis [4,5]), Russia (C. bescii [6,7]), C. kronotskyensis [2,8], and C. hydrothermalis [2,8]), New Zealand (C. saccharolyticus [9-11]), and Iceland (C. kristjanssonii [2,12] and C. lactoaceticus [2,13]). Characteristic of Caldicellulosiruptor species are multidomain extracellular and S-layer-associated glycoside hydrolases (GHs) that mediate the microbial conversion of complex carbohydrates (14-16). Sequenced genomes for Caldicellulosiruptor species (2, 4, 6, 10) indicate that some, but not all, encode GH48-containing enzymes (17), and these appear to be essential for crystalline cellulose degradation (18). The cellulolytic Caldicellulosiruptor species also utilize novel binding proteins (ta pirins) to adhere to plant biomass (19). Sever...

Section: T He Genusmentioning

confidence: 99%

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confidence: 99%

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization

Zurawski

Conway

Lee

et al. 2015

“…Examples of strictly anaerobic thermophiles that have been studied for biofuel production are Clostridium thermocellum (14,15), several Caldicellulosiruptor spp. (16)(17)(18), as well as Thermoanaerobacterium spp. and Thermoanaerobacter spp.…”

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confidence: 99%

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Bosma

Weijer

Daas

et al. 2015

b Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C 5 and C 6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216 T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 ؋ 10 3 colonies per g plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C 5 and C 6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals. Green chemicals are sustainable bio-based alternatives for chemicals based on fossil resources. Biomass is considered an attractive renewable resource for the production of such green chemicals. Major challenges for using biomass are to develop costeffective production processes and to convert substrates that go beyond first-generation pure sugars. From this perspective, the use of microbial fermentation processes that convert lignocellulosic sugars is gaining considerable attention. For particular products natural producers can be used, but often Escherichia coli and Saccharomyces cerevisiae are used as platform organisms because of their well-known physiology and ease of engineering for the production of many different products (1, 2).In general, most organisms used for the production of green chemicals and fuels are mesophiles, and current processes still are based mainly on first-generation feedstocks, i.e., sucrose from sugar beet or sugarcane or glucose derived from starches from corn or tapioca. Although the engineering of platform organisms broadens the spectrum of possible substrates (3), the efficiency of the process could be increased by the use of organisms naturally capable of degrading lignocellulose-derived sugars (4). To further increase the efficiency and reduce the costs of the microbial production of green chemicals, modera...

“…Southern blot analyses of C. bescii strains. Given the phenotypic abnormalities and observed IS element movements in the ΔpyrFA mutant (JWCB005) lineage strains described above, several strains were selected to probe for IS element movement via Southern blot analyses: JWCB005, JWCB018, JWCB032, and MACB1013 from the ΔpyrFA and MACB1013 are daughters of JWCB018 that were engineered for ethanol production using two different pathways, with JWCB032 having the highest ethanol yield of any strain of C. bescii to date (15). MACB1018 is a recently developed alternative genetic background strain that was used for the construction of MACB1032 and MACB1034, containing deletions of ldh and cbeI, respectively (5).…”

Section: Resultsmentioning

confidence: 99%

“…The deletion of the maturation genes required for the nickel-iron hydrogenase showed that this enzyme was not responsible for the majority of the hydrogen production by C. bescii (14). The addition of a bifunctional alcohol dehydrogenase gene (adhE) from Clostridium thermocellum, resulting in strain JWCB032, allowed for the production of ethanol from plant biomass at 65°C (15), and production at 75°C was obtained by expressing the genes encoding AdhE and AdhB from Thermoanaerobacter pseudethanolicus 39E, although the ethanol yield was much lower (16). JWCB032 is the best ethanol-producing strain of C. bescii to date, making it thus far the most promising strain for future industrial development.…”

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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

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.