Dataset Information

Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.

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 beta-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.

SUBMITTER: Zhou S

PROVIDER: S-EPMC92505 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.

Zhou S S Davis F C FC Ingram L O LO

Applied and environmental microbiology 20010101 1

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 beta- ...[more]

PMID: 11133422

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Project description:Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. Pectin is a complex polysaccharide. The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification. Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain. In addition to PaeY, the first pectin acetylesterase identified in the E. chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX. The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide. Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression. The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism. The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers. In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions. PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases. PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases. Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin. The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides. PaeX is mostly found in the periplasmic space of E. chrysanthemi. These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.

Project description:Erwinia chrysanthemi 3937 secretes several pectinolytic enzymes, among which eight isoenzymes of pectate lyases with an endo-cleaving mode (PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ) have been identified. Two exo-cleaving enzymes, the exopolygalacturonate lyase, PelX, and an exo-poly-alpha-D-galacturonosidase, PehX, have been previously identified in other E. chrysanthemi strains. Using a genomic bank of a 3937 mutant with the major pel genes deleted, we cloned a pectinase gene identified as pelX, encoding the exopolygalacturonate lyase. The deduced amino acid sequence of the 3937 PelX is very similar to the PelX of another E. chrysanthemi strain, EC16, except in the 43 C-terminal amino acids. PelX also has homology to the endo-pectate lyase PelL of E. chrysanthemi but has a N-terminal extension of 324 residues. The transcription of pelX, analyzed by gene fusions, is dependent on several environmental conditions. It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, nitrogen starvation, and catabolite repression. Regulation of pelX expression is dependent on the KdgR repressor, which controls almost all the steps of pectin catabolism, and on the global activator of sugar catabolism, cyclic AMP receptor protein. In contrast, PecS and PecT, two repressors of the transcription of most pectate lyase genes, are not involved in pelX expression. The pelX mutant displayed reduced pathogenicity on chicory leaves, but its virulence on potato tubers or Saintpaulia ionantha plants did not appear to be affected. The purified PelX protein has no maceration activity on plant tissues. Tetragalacturonate is the best substrate of PelX, but PelX also has good activity on longer oligomers. Therefore, the estimated number of binding subsites for PelX is 4, extending from subsites -2 to +2. PelX and PehX were shown to be localized in the periplasm of E. chrysanthemi 3937. PelX catalyzed the formation of unsaturated digalacturonates by attack from the reducing end of the substrate, while PehX released digalacturonates by attack from the nonreducing end of the substrate. Thus, the two types of exo-degrading enzymes appeared complementary in the degradation of pectic polymers, since they act on both extremities of the polymeric chain.

Dataset Information

Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.

Publications

Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2.

Similar Datasets

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