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Article ; Online: Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform.

Arnolds, Kathleen L / Higgins, Riley C / Crandall, Jennifer / Li, Gabriella / Linger, Jeffrey G / Guarnieri, Michael T

2023 Volume 87, Issue 1, Page(s) 12

Abstract: Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related ...

Abstract	Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae. We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.
MeSH term(s)	Ecosystem ; Saccharomyces cerevisiae/genetics ; Soil Microbiology ; Microbiota ; Soil ; Risk Assessment
Chemical Substances	Soil
Language	English
Publishing date	2023-12-11
Publishing country	United States
Document type	Journal Article
ZDB-ID	1462065-0
ISSN	1432-184X ; 0095-3628
ISSN (online)	1432-184X
ISSN	0095-3628
DOI	10.1007/s00248-023-02321-8
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Article ; Online: Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Brunecky, Roman / Knott, Brandon C / Subramanian, Venkataramanan / Linger, Jeffrey G / Beckham, Gregg T / Amore, Antonella / Taylor, Larry E / Vander Wall, Todd A / Lunin, Vladimir V / Zheng, Fei / Garrido, Mercedes / Schuster, Logan / Fulk, Emily M / Farmer, Samuel / Himmel, Michael E / Decker, Stephen R

The Journal of biological chemistry

2024 Volume 300, Issue 3, Page(s) 105749

Abstract: Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically ... ...

Abstract	Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.
MeSH term(s)	Aspergillus oryzae/enzymology ; Aspergillus oryzae/genetics ; Cellulose 1,4-beta-Cellobiosidase/chemistry ; Cellulose 1,4-beta-Cellobiosidase/classification ; Cellulose 1,4-beta-Cellobiosidase/genetics ; Cellulose 1,4-beta-Cellobiosidase/metabolism ; Enzyme Assays ; Genome, Fungal/genetics ; Mutation ; Protein Engineering/methods ; Substrate Specificity ; Talaromyces/enzymology ; Talaromyces/genetics ; Trichoderma/enzymology ; Trichoderma/genetics ; Trichoderma/metabolism ; Biocatalysis
Chemical Substances	Cellulose 1,4-beta-Cellobiosidase (EC 3.2.1.91) ; lignocellulose (11132-73-3)
Language	English
Publishing date	2024-02-13
Publishing country	United States
Document type	Comparative Study ; Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2024.105749
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Article ; Online: Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs.

Hernandez Hernandez, Diana / Ding, Lin / Murao, Ayako / Dahlin, Lukas R / Li, Gabriella / Arnolds, Kathleen L / Amezola, Melissa / Klein, Amit / Mitra, Aishwarya / Mecacci, Sonia / Linger, Jeffrey G / Guarnieri, Michael T / Suzuki, Yo

ACS synthetic biology

2023 Volume 12, Issue 9, Page(s) 2778–2782

Abstract: Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The ... ...

Abstract	Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.
MeSH term(s)	High-Throughput Nucleotide Sequencing ; DNA/genetics
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2023-08-15
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ISSN	2161-5063
ISSN (online)	2161-5063
DOI	10.1021/acssynbio.3c00183
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Article: Biotechnology for secure biocontainment designs in an emerging bioeconomy

Arnolds, Kathleen L / Dahlin, Lukas R / Ding, Lin / Wu, Chao / Yu, Jianping / Xiong, Wei / Zuniga, Cristal / Suzuki, Yo / Zengler, Karsten / Linger, Jeffrey G / Guarnieri, Michael T

Current opinion in biotechnology. 2021 Oct., v. 71

2021

Abstract: Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology ... ...

Abstract	Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.
Keywords	biocontainment ; bioeconomics ; bioenergy ; biosecurity ; biotechnology ; genome ; medicine ; synthetic biology
Language	English
Dates of publication	2021-10
Size	p. 25-31.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2021.05.004
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Article ; Online: Biotechnology for secure biocontainment designs in an emerging bioeconomy.

Arnolds, Kathleen L / Dahlin, Lukas R / Ding, Lin / Wu, Chao / Yu, Jianping / Xiong, Wei / Zuniga, Cristal / Suzuki, Yo / Zengler, Karsten / Linger, Jeffrey G / Guarnieri, Michael T

Current opinion in biotechnology

2021 Volume 71, Page(s) 25–31

Abstract	Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.
MeSH term(s)	Agriculture ; Biotechnology ; Ecosystem ; Genome ; Synthetic Biology
Language	English
Publishing date	2021-06-03
Publishing country	England
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2021.05.004
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Article ; Online: Conversion of levoglucosan and cellobiosan by

Linger, Jeffrey G / Hobdey, Sarah E / Franden, Mary Ann / Fulk, Emily M / Beckham, Gregg T

Metabolic engineering communications

2016 Volume 3, Page(s) 24–29

Abstract: Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of ...

Abstract	Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe
Language	English
Publishing date	2016-02-02
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2821894-2
ISSN	2214-0301 ; 2214-0301
ISSN (online)	2214-0301
ISSN	2214-0301
DOI	10.1016/j.meteno.2016.01.005
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Article: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440

Johnson, Christopher W / Abraham, Paul E / Linger, Jeffrey G / Khanna, Payal / Hettich, Robert L / Beckham, Gregg T

Metabolic Engineering Communications. 2017 Dec., v. 5

2017

Abstract: Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural ... ...

Abstract	Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals.
Keywords	Pseudomonas putida ; acetates ; carbon ; cell growth ; energy ; feedstocks ; genes ; glucose ; lignin ; mass spectrometry ; metabolic engineering ; microorganisms ; nylon ; proteomics ; value added
Language	English
Dates of publication	2017-12
Size	p. 19-25.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	2821894-2
ISSN	2214-0301
ISSN	2214-0301
DOI	10.1016/j.meteno.2017.05.002
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Article: Chromatin disassembly and reassembly during DNA repair.

Linger, Jeffrey G / Tyler, Jessica K

Mutation research

2007 Volume 618, Issue 1-2, Page(s) 52–64

Abstract: Current research is demonstrating that the packaging of the eukaryotic genome together with histone proteins into chromatin is playing a fundamental role in DNA repair and the maintenance of genomic integrity. As is well established to be the case for ... ...

Abstract	Current research is demonstrating that the packaging of the eukaryotic genome together with histone proteins into chromatin is playing a fundamental role in DNA repair and the maintenance of genomic integrity. As is well established to be the case for transcription, the chromatin structure dynamically changes during DNA repair. Recent studies indicate that the complete removal of histones from DNA and their subsequent reassembly onto DNA accompanies DNA repair. This review will present evidence indicating that chromatin disassembly and reassembly occur during DNA repair and that these are critical processes for cell survival after DNA repair. Concomitantly, candidate proteins utilized for these processes will be highlighted.
MeSH term(s)	Adenosine Triphosphate/chemistry ; Animals ; Chromatin/genetics ; Chromatin/metabolism ; Chromatin/physiology ; Chromatin Assembly and Disassembly ; DNA/chemistry ; DNA Repair ; Histones/chemistry ; Humans ; Models, Biological ; Models, Genetic ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Chromatin ; Histones ; Adenosine Triphosphate (8L70Q75FXE) ; DNA (9007-49-2)
Language	English
Publishing date	2007-05-01
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	206607-5
ISSN	1873-135X ; 0027-5107 ; 1383-5718 ; 0165-1110 ; 0165-1161 ; 0165-7992 ; 0921-8777 ; 0165-1218 ; 1383-5726 ; 0167-8817 ; 0921-8734 ; 1383-5742
ISSN (online)	1873-135X
ISSN	0027-5107 ; 1383-5718 ; 0165-1110 ; 0165-1161 ; 0165-7992 ; 0921-8777 ; 0165-1218 ; 1383-5726 ; 0167-8817 ; 0921-8734 ; 1383-5742
DOI	10.1016/j.mrfmmm.2006.05.039
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Article: Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

Linger, Jeffrey G / Hobdey, Sarah E / Franden, Mary Ann / Fulk, Emily M / Beckham, Gregg T

Metabolic Engineering Communications. 2016 Dec., v. 3

2016

Abstract	Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.
Keywords	Pseudomonas putida ; beta-glucosidase ; biofuels ; carbon ; cell walls ; cellobiose ; cellulose ; endo-1,4-beta-glucanase ; energy ; fractionation ; glucose ; glucose 6-phosphate ; glycosides ; polymers ; pyrolysis
Language	English
Dates of publication	2016-12
Size	p. 24-29.
Publishing place	Elsevier B.V.
Document type	Article
ZDB-ID	2821894-2
ISSN	2214-0301
ISSN	2214-0301
DOI	10.1016/j.meteno.2016.01.005
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Article ; Online: Heterologous expression and extracellular secretion of cellulolytic enzymes by Zymomonas mobilis.

Linger, Jeffrey G / Adney, William S / Darzins, Al

Applied and environmental microbiology

2010 Volume 76, Issue 19, Page(s) 6360–6369

Abstract: Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the ...

Abstract	Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.
MeSH term(s)	Actinomycetales/enzymology ; Actinomycetales/genetics ; Biotechnology/methods ; Carboxymethylcellulose Sodium/metabolism ; Cellulases/genetics ; Cellulases/metabolism ; DNA, Bacterial/chemistry ; DNA, Bacterial/genetics ; Gene Expression ; Molecular Sequence Data ; Protein Sorting Signals/genetics ; Protein Transport ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Sequence Analysis, DNA ; Zymomonas/enzymology ; Zymomonas/genetics ; Zymomonas/metabolism
Chemical Substances	DNA, Bacterial ; Protein Sorting Signals ; Recombinant Fusion Proteins ; Cellulases (EC 3.2.1.-) ; Carboxymethylcellulose Sodium (K679OBS311)
Language	English
Publishing date	2010-08-06
Publishing country	United States
Document type	Journal Article
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/AEM.00230-10
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