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  • 1
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO2 and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO2. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.
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  • 2
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. In cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640 mU/mg), phosphoribose isomerase (1600 mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO2 and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly.
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  • 3
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    Springer
    Archives of microbiology 164 (1995), S. 324-330 
    ISSN: 1432-072X
    Keywords: Key wordsEscherichia coli ; 6-Phosphogluconate ; dehydrogenase ; Ribulose-5-phosphate epimerase ; Ribose-5-phosphate isomerase ; Transketolase ; Transaldolase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The pentose-phosphate pathway of Escherichia coli K-12, in addition to its role as a route for the breakdown of sugars such as glucose or pentoses, provides the cell with intermediates for the anabolism of amino acids, vitamins, nucleotides, and cell wall constituents. Through its oxidative branch, it is a major source of NADPH. The expression of the gene for NADP-dependent 6-phosphogluconate dehydrogenase (gnd) is regulated by the growth rate in E. coli. The recently identified gene for ribulose-5-phosphate 3-epimerase (rpe) is part of a large operon that comprises among others genes for the biosynthesis of aromatic amino acids. In recent years, genes for all enzymes of the pathway have been cloned and sequenced. Isoenzymes have been found for transketolase (genes tktA and tktB), ribose-5-phosphate isomerase (rpiA and rpiB) and transaldolase (talA and talB).
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  • 4
    ISSN: 1432-072X
    Keywords: d-Fructose ; New pathway ; l-Sorbose ; E. coli ; K. pneumoniae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Starting with a fruK (formerly fpk) mutant of Escherichia coli K12 lacking d-fructose-1-phosphate kinase (E.C. 2.7.1.3.), fructose positive derivatives were isolated after introduction of the cloned gene sorE from Klebsiella pneumoniae coding for an l-sorbose-1-phosphate reductase. The new pathway was shwon to proceed from d-fructose via d-fructose-1-phosphate and d-mannitol-1-phosphate to d-fructose 6-phosphate. It involves a transport system and enzymes encoded in the fru and the mtl operons from E. coli K12 as well as in the sor operon from K. pneumoniae respectively.
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  • 5
    ISSN: 1432-072X
    Keywords: Key wordsZymomonas mobilis ; Metabolic flux ; analysis ; Sugar phosphates ; Glucose ; Fructose ; Xylose ; 13C-NMR ; In vivo 31P-NMR ; Rate-limiting step
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium Zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. A xylose-fermenting recombinant strain of Z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and a strongly reduced growth rate as compared to the parent strain. Therefore, a comprehensive study employing recently developed NMR-based methods for the in vivo analysis of intracellular phosphorylated pool sizes and metabolic fluxes was undertaken to enable a global characterization of the intracellular metabolic state of Z. mobilis during growth on 13C-labelled glucose, fructose and xylose in defined continuous cultures. The 13C-NMR flux analysis indicated that ribose 5-phosphate is synthesized via the nonoxidative pentose phosphate pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the level of heterologous xylulokinase. The 31P-NMR analyses revealed a global alteration of the levels of intracellular phosphorylated metabolites during growth on fructose as compared to that on glucose. The results suggest that this is primarily caused by an elevated concentration of intracellular fructose 6-phosphate.
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  • 6
    ISSN: 1433-8491
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
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  • 7
    ISSN: 1617-4623
    Keywords: sor genes ; l-sorbose ; Chromosomal rearrangement ; Klebsiella pneumoniae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.
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  • 8
    ISSN: 1617-4623
    Keywords: Klebsiella pneumoniae ; λplac-Mu hybrids ; LacZ fusions ; Hfr strains ; Conjugation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Klebsiella pneumoniae 1033-5P14 and its P1-sensitive derivative KAY2026 were found to be resistant to λ although they contained a LamB protein, active as a maltoporin. Sensitive derivatives could only be obtained after introduction of the pTROY9 plasmid which expresses lamB and the corresponding λ receptor from Escherichia coli K12 at high levels. Lysogenic derivatives from such strains were shown to carry the phage at secondary att sites and to give high titer lysates when induced. The use of λplac-Mu hybrid phages allowed the isolation from several operons of lacZ fusions orientated in, or against, the direction of transcription. Such insertions could subsequently be used to isolate stable Hfr strains by allowing homologous recombination to take place between the lac genes in the inserted hybrid phages and those of plasmid F′ts114 lac+ zzf20::Tn10. The Hfr strains were able to transfer K. pneumoniae chromosomal genes and allowed the mapping of such genes. Characteristic differences between this conjugation system and that of Escherichia coli K12 are discussed. The insertions also allowed determination of the direction of transcription of the gut gene, the newly mapped scr gene and of the sor gene cluster encoding enzymes for the metabolism of d-glucitol, sucrose and l-sorbose.
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  • 9
    ISSN: 1617-4623
    Keywords: Klebsiella pneumoniae ; Xylose isomerase ; Xylulokinase ; xy1 gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The genes xy1A and xy1B were cloned together with their promoter region from the chromosome of Klehsiella pneumoniae var. aerogenes 1033 and the DNA sequence (3225 bp) was determined. The gene xy1A encodes the enzyme xylose isomerase (XI or XylA) consisting of 440 amino acids (calculated Mr of 49 793). The gene xy1B encodes the enzyme xylulokinase (XK or Xy1B) with a calculated M, of 51 783 (483 amino acids). The two genes successfully complemented xy1 mutants of Escherichia coli K12, but no gene dosage effect was detected. E. coli wild-type cells which harbored plasmids with the intact xylA Kp 5′ upstream region in high copy number (but lacking an active xy1B gene on the plasmids) were phenotypically xylose-negative and xylose isomerase and xylulokinase activities were drastically diminished. Deletion of 5′ upstream regions of xy1A on these plasmids and their substitution by a lac promoter resulted in a xylose-positive phenotype. This also resulted in overproduction of plasmid-encoded xylose isomerase and xylulokinase activities in recombinant E. coli cells.
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  • 10
    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.
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