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  • Biology  (4)
  • 1
    Online Resource
    Online Resource
    Functional Replacement of the Escherichia coli d- (−)-Lactate Dehydrogenase Gene ( ldhA ) with the l- (+)-Lactate Dehydrogenase Gene ( ldhL) from Pediococcus acidilactici
    American Society for Microbiology ; 2003
    In:  Applied and Environmental Microbiology Vol. 69, No. 4 ( 2003-04), p. 2237-2244
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 69, No. 4 ( 2003-04), p. 2237-2244
    Abstract: The microbial production of l- (+)-lactic acid is rapidly expanding to allow increased production of polylactic acid (PLA), a renewable, biodegradable plastic. The physical properties of PLA can be tailored for specific applications by controlling the ratio of l- (+) and d -(−) isomers. For most uses of PLA, the l- (+) isomer is more abundant. As an approach to reduce costs associated with biocatalysis (complex nutrients, antibiotics, aeration, product purification, and waste disposal), a recombinant derivative of Escherichia coli W3110 was developed that contains five chromosomal deletions ( focA-pflB frdBC adhE ackA ldhA ). This strain was constructed from a d -(−)-lactic acid-producing strain, SZ63 ( focA-pflB frdBC adhE ackA ), by replacing part of the chromosomal ldhA coding region with Pediococcus acidilactici ldhL encoding an l -lactate dehydrogenase. Although the initial strain (SZ79) grew and fermented poorly, a mutant (SZ85) was readily isolated by selecting for improved growth. SZ85 exhibited a 30-fold increase in l -lactate dehydrogenase activity in comparison to SZ79, functionally replacing the native d -lactate dehydrogenase activity. Sequencing revealed mutations in the upstream, coding, and terminator regions of ldhL in SZ85, which are presumed to be responsible for increased l -lactate dehydrogenase activity. SZ85 produced l -lactic acid in M9 mineral salts medium containing glucose or xylose with a yield of 93 to 95%, a purity of 98% (based on total fermentation products), and an optical purity greater than 99%. Unlike other recombinant biocatalysts for l -lactic acid, SZ85 remained prototrophic and is devoid of plasmids and antibiotic resistance genes.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.69.4.2237-2244.2003
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 2
    Online Resource
    Online Resource
    Enhancement of Expression and Apparent Secretion of Erwinia chrysanthemi Endoglucanase (Encoded by celZ ) in Escherichia coli B
    American Society for Microbiology ; 1999
    In:  Applied and Environmental Microbiology Vol. 65, No. 6 ( 1999-06), p. 2439-2445
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 65, No. 6 ( 1999-06), p. 2439-2445
    Abstract: Escherichia coli B has been engineered as a biocatalyst for the conversion of lignocellulose into ethanol. Previous research has demonstrated that derivatives of E. coli B can produce high levels of Erwinia chrysanthemi endoglucanase (encoded by celZ ) as a periplasmic product and that this enzyme can function with commercial fungal cellulase to increase ethanol production. In this study, we have demonstrated two methods that improve celZ expression in E. coli B. Initially, with a low-copy-number vector, two E. coli glycolytic gene promoters ( gap and eno ) were tested and found to be less effective than the original celZ promoter. By screening 18,000 random fragments of Zymomonas mobilis DNA, a surrogate promoter was identified which increased celZ expression up to sixfold. With this promoter, large polar inclusion bodies were clearly evident in the periplasmic space. Sequencing revealed that the most active surrogate promoter is derived from five Sau 3A1 fragments, one of which was previously sequenced in Z. mobilis . Visual inspection indicated that this DNA fragment contains at least five putative promoter regions, two of which were confirmed by primer extension analysis. Addition of the out genes from E. chrysanthemi EC16 caused a further increase in the production of active enzyme and facilitated secretion or release of over half of the activity into the extracellular environment. With the most active construct, of a total of 13,000 IU of active enzyme per liter of culture, 7,800 IU was in the supernatant. The total active endoglucanase was estimated to represent 4 to 6% of cellular protein.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.65.6.2439-2445.1999
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 1999
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 3
    Online Resource
    Online Resource
    Production of Optically Pure d -Lactic Acid in Mineral Salts Medium by Metabolically Engineered Escherichia coli W3110
    American Society for Microbiology ; 2003
    In:  Applied and Environmental Microbiology Vol. 69, No. 1 ( 2003-01), p. 399-407
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 69, No. 1 ( 2003-01), p. 399-407
    Abstract: The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of l -lactic acid to d -lactic acid. Although the largest demand is for the l enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of d -lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. d -Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this d -lactic acid was ∼98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase ( frdABCD ), alcohol/aldehyde dehydrogenase ( adhE ), and pyruvate formate lyase ( pflB ). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene ( ackA ). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of d -lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.69.1.399-407.2003
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 4
    Online Resource
    Online Resource
    Gene Integration and Expression and Extracellular Secretion of Erwinia chrysanthemi Endoglucanase CelY ( celY ) and CelZ ( celZ ) in Ethanologenic Klebsiella oxytoca P2
    American Society for Microbiology ; 2001
    In:  Applied and Environmental Microbiology Vol. 67, No. 1 ( 2001-01), p. 6-14
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 67, No. 1 ( 2001-01), p. 6-14
    Abstract: The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.67.1.6-14.2001
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
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