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  • 1
    ISSN: 1432-072X
    Keywords: Key words Glucose-fructose oxidoreductase ; Zymomonas mobilis ; Gfo-deficient mutant ; Sorbitol ; Protein export ; Signal sequence ; Periplasmic NADP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32–46; Δ32–46) led to a protein that was no longer processed, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence (residues 2–20; Δ2–20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Δ2–20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Δ32–46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1617-4623
    Keywords: Pyruvate decarboxylase gene (pdc) ; Expression vectors ; Promoter/terminator ; Chloramphenicol acetyltransferase ; Zymomonas mobilis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A set of vectors was constructed for the cloning and expression of heterologous genes in the Gramnegative bacterium Zymomonas mobilis under the control of the pdc promoter of Z. mobilis. The vectors pPTZ1, pPTZ3, and pPTZ4 are based on the cryptic Z. mobilis plasmid pZM02 and on parts of the Escherichia coli plasmids pKK223-3 and pBR322 together with the multiple cloning site of phage Ml3mp18. DNA fragments can be readily inserted immediately downstream from the pdc promoter at unique restriction sites for KpnI, XbaI and PstI in pPTZl and additionally for SmaI and BamHI in pPTZ3. In pPTZ4, the 5′ terminal codons of pdc were deleted allowing the formation of gene fusions. Expression of a promoterless chloramphenicol acetyltransferase gene (cat) controlled by the pdc gene promoter resulted in enzyme activities of up to 5.5 U/mg total cell protein in Z. mobilis cells.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-0972
    Keywords: Cloning vectors ; ethanol ; plasmids ; strain improvement ; Zymomonas genetics ; Zymomonas mobilis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Present knowledge on the genetics of the ethanologenic anaerobeZymomonas mobilis includes background information on: size, restriction, and to some extent hybridization, analysis of indigenous plasmids; mutagenesis and isolation of a wide variety of auxotrophic, drug resistant and conditional mutants; construction of shuttle cloning vectors able to replicate and express inZ. mobilis; development of gene transfer systems based on conjugal mobilization of plasmids fromEscherichia coli donors toZ. mobilis; expression of heterologous genes inZ. mobilis; cloning and analysis of genes encoding enzymes of the Entner-Doudoroff pathway. Moreover, preliminary data on recombinational repair mechanisms and plasmid stability, which are now available, makeZ. mobilis an attractive model system for molecular genetic research and, furthermore, they contribute towards expansion of the substrate and product range of this industrial microorganism.
    Type of Medium: Electronic Resource
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