Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Feldmann, Sigrun D.; Sahm, Hermann; Sprenger, Georg A.

doi:10.1007/bf00283840

Cited by 26 publications

(14 citation statements)

References 49 publications

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“…XK was assayed by measuring xylulose-5-phosphate formation in a coupled reaction catalyzed by pyruvate kinase and lactate dehydrogenase (8). The assay mixture contained 56 mM Tris-HCl, 56 mM KCl (pH 7.8), 1 mM EDTA, 5.6 mM MgCl 2 , 1 mM phosphoenol pyruvate, 0.5 mM ATP, 0.3 mM NADH, 0.3 U lactate dehydrogenase, 0.3 U pyruvate kinase, and cell extract in a total volume of 900 l. The reaction was started by adding 100 l of 10 mM D-xylulose, after which NADH oxidation was determined spectroscopically as absorbance at 340 nm recorded at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Yanase

Sato

Yamamoto

et al. 2007

Appl Environ Microbiol

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Its metabolic characteristics suggest that Zymobacter palmae gen. nov., sp. nov. could serve as a useful new ethanol-fermenting bacterium, but its biotechnological exploitation will require certain genetic modifications. We therefore engineered Z. palmae so as to broaden the range of its fermentable sugar substrates to include the pentose sugar xylose. The Escherichia coli genes encoding the xylose catabolic enzymes xylose isomerase, xylulokinase, transaldolase, and transketolase were introduced into Z. palmae, where their expression was driven by the Zymomonas mobilis glyceraldehyde-3-phosphate dehydrogenase promoter. When cultured with 40 g/liter xylose, the recombinant Z. palmae strain was able to ferment 16.4 g/liter xylose within 5 days, producing 91% of the theoretical yield of ethanol with no accumulation of organic acids as metabolic by-products. Notably, xylose acclimation enhanced both the expression of xylose catabolic enzymes and the rate of xylose uptake into recombinant Z. palmae, which enabled the acclimated organism to completely and simultaneously ferment a mixture of 40 g/liter glucose and 40 g/liter xylose within 8 h, producing 95% of the theoretical yield of ethanol. Thus, efficient fermentation of a mixture of glucose and xylose to ethanol can be accomplished by using Z. palmae expressing E. coli xylose catabolic enzymes.

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Section: Methodsmentioning

confidence: 99%

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Yanase

Sato

Yamamoto

et al. 2007

Appl Environ Microbiol

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“…XI and XK were assayed discontinuously as described before (Feldmann et al 1992). The XI assay measured xylulose (formed from xylose) with sorbitol dehydrogenase.…”

Section: Methodsmentioning

confidence: 99%

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

Feldmann

Sahm

Sprenger

1992

Appl Microbiol Biotechnol

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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. I n cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640mU/mg), phosphoribose isomerase (1600mU/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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“…The active-site residues and residues mediating subunit interactions in the group I XIs are conserved in the group II XIs (2,14,25,32). The active-site residues of the Streptomyces rubiginosus XI (38) are conserved through the 20 group I and II XIs including the 210 and IO-1 XIs (Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…Isomerization and phosphorylation are catalyzed by D-xylose isomerase (XI; encoded by xylA) and D-xylulose kinase (encoded by xylB), respectively. In most cases, these genes are clustered into an operon (xyl operon) (14,22,24,33,39).…”

mentioning

confidence: 99%

“…The average length of their polypeptide chains is 380 to 390 amino acids, and they have about 60% amino acid sequence identity, with the active-site residues being highly conserved. The enzymes from Klebsiella (14), Escherichia (22,34), Lactobacillus (3,4,24,40), Lactococcus (13), Clostridium (25), Bacillus (28), Staphylococcus (33), and Thermoanaerobacter (23) are classified in group II. They are 440 to 460 amino acids long and show more than 50% amino acid sequence identity among the group members.…”

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confidence: 99%

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Restoration of a Defective Lactococcus lactis Xylose Isomerase

Park

Batt

2004

Appl Environ Microbiol

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The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl ؊ ) and IO-1 (Xyl ؉ ), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K m and k cat being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K m and k cat of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

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Cloning and expression of the genes for xylose isomerase and xylulokinase from Klebsiella pneumoniae 1033 in Escherichia coli K12

Cited by 26 publications

References 49 publications

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Genetic Engineering of Zymobacter palmae for Production of Ethanol from Xylose

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

Restoration of a Defective Lactococcus lactis Xylose Isomerase

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