Characterization of the gene celD and its encoded product 1,4-β-<scp>d</scp>-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa

Rixon, Jane E.; Ferreira, L.M.A.; Durrant, A. J.; Laurie, Judith I.; Hazlewood, Geoffrey P.; Gilbert, Harry J.

doi:10.1042/bj2850947

Cited by 47 publications

(34 citation statements)

References 34 publications

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“…To further hydrolyze cellodextrins into glucose, we screened four β-glucosidases cloned from Cellvibrio japonicus, a Gram-negative cellulolytic bacterium, and determined if their expression in E. coli could permit growth on cellobiose (Fig. 1B) (29,30). E. coli grew best on cellobiose when expressing either cel3A or cel3B (Fig.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

Bokinsky

Peralta‐Yahya

George

et al. 2011

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

342

263

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One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli , to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.

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Section: Resultsmentioning

confidence: 99%

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

Bokinsky

Peralta‐Yahya

George

et al. 2011

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

342

263

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“…The endogluconases exhibit a general hydrolytic activity for internal glycosidic linkages with a consequential increase in the reducing sugar concentration and decrease in the polymer length [81,82]. Furthermore, the action of glucan glucanohydrolases was reported on cello-oligomers with glucose production as a primary product [83,84]. However, the action of the GH-12 cellulase of Thermotoga neapolitana on cellotriose and cellotretrose resulted in the production of small quantities of glucose after prolonged periods of incubation with cellobiose as the main product [85].…”

Section: Discussionmentioning

confidence: 99%

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

Ibrahim¹,

Jones²,

Hossenya³

2013

J Pet Environ Biotechnol

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Lignocellulosic biomass has potential for bioethanol, a renewable fuel. A limitation is that bioconversion of the complex lignocellulosic material to simple sugars and then to bioethanol is a challenging process. Recent work has focused on the genetic engineering of a biocatalyst that may play a critical role in biofuel production. Escherichia coli have been considered a convenient host for biocatalysts in biofuel production for its fermentation of glucose into a wide range of short-chain alcohols and production of highly deoxygenated hydrocarbon. The bacterium Pectobacterium carotovorum subsp. carotovorum (P. carotovorum) is notorious for its maceration of the plant cell wall causing soft rot. The ability to destroy plants is due to the expression and secretion of a wide range of hydrolytic enzymes that include cellulases and polygalacturonases. P. carotovorum ATCC™ no. 15359 was used as a source of DNA for the amplification of celB, celC and peh. These genes encode 2 cellulases and a polygalacturonase, respectively. Primers were designed based on published gene sequences and used to amplify the open reading frames from the genomic DNA of P. carotovorum. The individual PCR products were cloned into the pTAC-MAT-2 expression vector and transformed into Escherichia coli. The deduced amino acid sequences of the cloned genes have been analyzed for their catalytically active domains. Estimation of the molecular weights of the expressed proteins was performed using SDS-PAGE analysis and celB, celC and peh products were approximately 29.5 kDa, 40 kDa 41.5 kDa, respectively. Qualitative determination of the cellulase and polygalacturonase activities of the cloned genes was carried out using agar diffusion assays.

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“…Pseudomonas fluorescens subsp. cellulosa was also shown to constitutively express polysaccharidases (Rixon et al, 1992), although their role in the regulation of gene expression in the pseudomonad is currently unknown.…”

Section: Abbreviationmentioning

confidence: 99%

A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions The GenBank accession numbers for the sequences described in this paper are AF049493 and AF168359 for xynC and xynG, respectively.

Fontes¹,

Gilbert

Hazlewood

et al. 2000

Microbiology

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Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylooligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in λZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M r of 43 413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG ; XylG and xynG were 67 % and 63 % identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M r of 41 000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.

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Characterization of the gene celD and its encoded product 1,4-β-d-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa

Cited by 47 publications

References 34 publications

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli

Molecular Cloning and Expression of Cellulase and Polygalacturonase Genes in E. coli as a Promising Application for Biofuel Production

A novel Cellvibrio mixtus family 10 xylanase that is both intracellular and expressed under non-inducing conditions The GenBank accession numbers for the sequences described in this paper are AF049493 and AF168359 for xynC and xynG, respectively.

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