Pectobacterium atrosepticum KDPG aldolase, Eda, participates in the Entner-Doudoroff pathway and independently inhibits expression of virulence determinants.

Pectobacterium carotovorum has an incomplete Entner-Doudoroff (ED) pathway, including enzyme 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda) but lacking phosphogluconate dehydratase (Edd), while P. atrosepticum (Pba) has a complete pathway. To understand the role of the ED pathway in Pectobacterium infection, mutants of these two key enzymes, Δeda and Δedd, were constructed in Pba SCRI1039. Δeda exhibited significant decreased virulence on potato tubers and colonization in planta and was greatly attenuated in pectinase activity and the ability to use pectin breakdown products, including polygalacturonic acid (PGA) and galacturonic acid. These reduced phenotypes were restored following complementation with an external vector expressing eda. Quantitative reverse transcription PCR analysis revealed that expression of the pectinase genes pelA, pelC, pehN, pelW, and pmeB in Δeda cultured in pyruvate, with or without PGA, was significantly reduced compared to the wild type, while genes for virulence regulators (kdgR, hexR, hexA, and rsmA) remained unchanged. However, Δedd showed similar phenotypes to the wild type. To our knowledge, this is the first demonstration that disruption of eda has a feedback effect on inhibiting pectin degradation and that Eda is involved in building the arsenal of pectinases needed during infection by Pectobacterium.

Vitamin B6 prevents excessive inflammation by reducing accumulation of sphingosine-1-phosphate in a sphingosine-1-phosphate lyase-dependent manner.

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti-inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti-inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine-1-phosphate (S1P) in a S1P lyase (SPL)-dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro-inflammatory cytokines by suppressing nuclear factor-κB and mitogen-activated protein kinases signalling pathways. Furthermore, vitamin B6-reduced accumulation of S1P by promoting SPL activity. The anti-inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti-inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.

Responsiveness of sphingosine phosphate lyase insufficiency syndrome to vitamin B6 cofactor supplementation.

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation.

Functional aptitude of hake minces with added TMAO-demethylase inhibitors during frozen storage.

The ability of compounds of natural origin (black, white, red, and green tea extracts, phytic acid) to inhibit TMAO-demethylase enzyme was assayed. Black tea and phytic acid exerted the highest inhibiting activities, similar to the already known inhibitor sodium citrate. Hake minces incorporating these three compounds were prepared and stored frozen (150 days, -12 °C). TMAO-demethylase enzyme was partially inhibited (lower enzyme activity, reduction of formaldehyde accumulation). The study of physicochemical properties of the minces (salt-soluble proteins, water holding capacity, structural water associated with myofibrils) pointed to evident protein aggregation and loss of functionality when phytic acid was added, whereas black tea and sodium citrate did not have a negative effect. Consequently, the salt-ground mince with phytic acid showed worse viscoelastic properties than the others. In conclusion, black tea polyphenols and sodium citrate can be used as additives to inhibit TMAO-demethylase enzyme during frozen storage of fish minces.

Recombinant Lipoxygenases and Hydroperoxide Lyases for the Synthesis of Green Leaf Volatiles.

Green leaf volatiles (GLVs) are mainly C6- and in rare cases also C9-aldehydes, -alcohols, and -esters, which are released by plants in response to biotic or abiotic stresses. These compounds are named for their characteristic smell reminiscent of freshly mowed grass. This review focuses on GLVs and the two major pathway enzymes responsible for their formation: lipoxygenases (LOXs) and fatty acid hydroperoxide lyases (HPLs). LOXs catalyze the peroxidation of unsaturated fatty acids, such as linoleic and α-linolenic acids. Hydroperoxy fatty acids are further converted by HPLs into aldehydes and oxo-acids. In many industrial applications, plant extracts have been used as LOX and HPL sources. However, these processes are limited by low enzyme concentration, stability, and specificity. Alternatively, recombinant enzymes can be used as biocatalysts for GLV synthesis. The increasing number of well-characterized enzymes efficiently expressed by microbial hosts will foster the development of innovative biocatalytic processes for GLV production.

Nicotinamide phosphoribosyltransferase is a molecular target of potent anticancer agents identified from phenotype-based drug screening.

Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.

Structure-Based Virtual Screening and Biochemical Evaluation for the Identification of Novel Trypanosoma brucei Aldolase Inhibitors.

INTRODUCTION: The glycolytic enzyme fructose-1,6-bisphosphate aldolase is a validated molecular target in human African trypanosomiasis (HAT) drug discovery, a neglected tropical disease (NTD) caused by the protozoan Trypanosoma brucei. Herein, a structure-based virtual screening (SBVS) approach to the identification of novel T. brucei aldolase inhibitors is described. Distinct molecular docking algorithms were used to screen more than 500,000 compounds against the X-ray structure of the enzyme. This SBVS strategy led to the selection of a series of molecules which were evaluated for their activity on recombinant T. brucei aldolase. The effort led to the discovery of structurally new ligands able to inhibit the catalytic activity of the enzyme. RESULTS: The predicted binding conformations were additionally investigated in molecular dynamics simulations, which provided useful insights into the enzyme-inhibitor intermolecular interactions. CONCLUSION: The molecular modeling results along with the enzyme inhibition data generated practical knowledge to be explored in further structure-based drug design efforts in HAT drug discovery.

Olive Recombinant Hydroperoxide Lyase, an Efficient Biocatalyst for Synthesis of Green Leaf Volatiles.

Volatile C6-aldehydes are the main contributors to the characteristic odor of plants known as "green note" and are widely used by the flavor industry. Biotechnological processes were developed to fulfill the high demand in C6-aldehydes in natural flavorants and odorants. Recombinant hydroperoxide lyases (HPLs) constitute an interesting alternative to overcome drawbacks arising from the use of HPL from plant extracts. Thus, olive recombinant 13-HPL was assayed as biocatalysts to produce C6-aldehydes. Firstly, a cDNA encoding for olive HPL of Leccino variety was isolated and cloned in pQE-30 expression vector. In order to improve the enzyme solubility, its chloroplast transit peptide was deleted. Both enzymes (HPL wild type and HPL deleted) were expressed into Escherichia coli strain M15, purified, characterized, and then used for bioconversion of 13-hydroperoxides of linoleic and linolenic acids. Aldehydes produced were extracted, then identified and quantified using gas chromatography and mass spectrometry. Recombinant HPL wild type (HPLwt) allowed producing 5.61 mM of hexanal and 4.39 mM of 3Z-hexenal, corresponding to high conversion yields of 93.5 and 73 %, respectively. Using HPL deleted (HPLdel) instead of HPLwt failed to obtain greater quantities of hexanal or 3Z-hexenal. No undesirable products were formed, and no isomerization of 3Z-hexenal in 2E-hexenal occurred. The olive recombinant HPLwt appears to be a promising efficient biocatalyst for the production of C6-aldehydes.

Molecular Cloning and Characterization of Hydroperoxide Lyase Gene in the Leaves of Tea Plant (Camellia sinensis).

Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST searches showed high homology to HPL sequences from other plants. The His-tag expression vector pET-32a(+)/CsHPL was constructed and transferred into Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E. coli was about 60 kDa. The enzyme activity of CsHPL was 0.20 µmol·min(-1)·mg(-1). Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea plants after Ectropis obliqua attack, suggesting that it may be an important candidate for defense against insects in tea plants.

CYP74B24 is the 13-hydroperoxide lyase involved in biosynthesis of green leaf volatiles in tea (Camellia sinensis).

Green leaf volatiles (GLVs) are C6-aliphatic aldehydes/alcohols/acetates, and biosynthesized from the central precursor fatty acid 13-hydroperoxides by 13-hydroperoxide lyases (HPLs) in various plant species. While GLVs have been implicated as defense compounds in plants, GLVs give characteristic grassy note to a bouquet of aroma in green tea, which is manufactured from young leaves of Camellia sinensis. Here we identify three HPL-related genes from C. sinensis via RNA-Sequencing (RNA-Seq) in silico, and functionally characterized a candidate gene, CYP74B24, as a gene encoding tea HPL. Recombinant CYP74B24 protein heterologously expressed in Escherichia coli specifically produced (Z)-3-hexenal from 13-HPOT with the optimal pH 6.0 in vitro. CYP74B24 gene was expressed throughout the aerial organs in a rather constitutive manner and further induced by mechanical wounding. Constitutive expression of CYP74B24 gene in intact tea leaves might account for low but substantial and constitutive formation of a subset of GLVs, some of which are stored as glycosides. Our results not only provide novel insights into the biological roles that GLVs play in tea plants, but also serve as basis for the improvement of aroma quality in tea manufacturing processes.

Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for n-butanol production.

BACKGROUND: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. RESULTS: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. CONCLUSION: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis.

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various ß-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the ß-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

The ssbL gene harbored by the ColV plasmid of an Escherichia coli neonatal meningitis strain is an auxiliary virulence factor boosting the production of siderophores through the shikimate pathway.

The ability to capture iron is a challenge for most bacteria. The neonatal meningitis Escherichia coli strain S88 possesses several iron uptake systems, notably including siderophores. Transcriptional analysis of the ColV plasmid pS88 has shown strong induction of a previously undescribed gene with low identity to three E. coli chromosomal genes encoding phospho-2-dehydro-3-deoxyheptonate aldolases involved in aromatic amino acid and catecholate/phenolate siderophore biosynthesis through the shikimate pathway. Here, we investigated the role of this gene, ssbLp (ssbL carried on the plasmid), in siderophore biosynthesis and, consequently, in S88 virulence. We constructed an S88 mutant designated S88 ΔssbLp, which exhibited reduced growth under low-iron conditions compared to the wild-type strain. Liquid chromatography-mass spectroscopy analysis of culture supernatants showed that the mutant secreted significantly smaller amounts of enterobactin, salmochelin SX, and yersiniabactin than the wild-type strain. The mutant was also less virulent in a neonatal rat sepsis model, with significantly lower bacteremia and mortality. Supplementation with chorismate, the final product of the shikimate pathway, restored the wild-type phenotype in vitro. In a collection of human extraintestinal E. coli isolates, we found that ssbL was present only in strains harboring the iro locus, encoding salmochelins, and was located either on the chromosome or on plasmids. Acquisition of the iro locus has been accompanied by acquisition of the auxiliary gene ssbL, which boosts the metabolic pathway essential for catecholate/phenolate siderophore biosynthesis and could represent potential therapeutic targets.

Metabolic profiling reveals sphingosine-1-phosphate kinase 2 and lyase as key targets of (phyto-) estrogen action in the breast cancer cell line MCF-7 and not in MCF-12A.

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.

Overexpression of hydroperoxide lyase, peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors.

Plants produce short-chain aldehydes and hydroxy fatty acids, which are important industrial materials, through the lipoxygenase pathway. Based on the information that lipoxygenase activity is up-regulated in tobacco leaves upon infection with tobacco mosaic virus (TMV), we introduced a melon hydroperoxide lyase (CmHPL) gene, a tomato peroxygenase (SlPXG) gene and a potato epoxide hydrolase (StEH) into tobacco leaves using a TMV-based viral vector system to afford aldehyde and hydroxy fatty acid production. Ten days after infiltration, tobacco leaves infiltrated with CmHPL displayed high enzyme activities of 9-LOX and 9-HPL, which could efficiently transform linoleic acid into C(9) aldehydes. Protein extracts prepared from 1 g of CmHPL-infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of control vector-infiltrated tobacco leaves (as an additional 9-LOX source) produced 758 ± 75 µg total C(9) aldehydes in 30 min. The yield of C(9) aldehydes from linoleic acid was 60%. Besides, leaves infiltrated with SlPXG and StEH showed considerable enzyme activities of 9-LOX/PXG and 9-LOX/EH, respectively, enabling the production of 9,12,13-trihydroxy-10(E)-octadecenoic acid from linoleic acid. Protein extracts prepared from 1 g of SlPXG-infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of StEH-infiltrated tobacco leaves produced 1738 ± 27 µg total 9,12,13-trihydroxy-10(E)-octadecenoic acid isomers in 30 min. The yield of trihydroxyoctadecenoic acids from linoleic acid was 58%. C(9) aldehydes and trihydroxy fatty acids could likely be produced on a larger scale using this expression system with many advantages including easy handling, time-saving and low production cost.

Thermal inactivation kinetics of recombinant proteins of the lipoxygenase pathway related to the synthesis of virgin olive oil volatile compounds.

The aim of this work was to characterize the thermal inactivation parameters of recombinant proteins related to the biosynthesis of virgin olive oil (VOO) volatile compounds through the lipoxygenase (LOX) pathway. Three purified LOX isoforms (Oep2LOX1, Oep1LOX2, and Oep2LOX2) and a hydroperoxide lyase (HPL) protein (OepHPL) were studied. According to their thermal inactivation parameters, recombinant Oep1LOX2 and Oep2LOX2 could be identified as the two LOX isoforms active in olive fruit crude preparations responsible for the synthesis of 13-hydroperoxides, the main substrates for the synthesis of VOO volatile compounds. Recombinant Oep2LOX1 displayed a low thermal stability, which suggests a weak actuation during the oil extraction process considering the current thermal conditions of this industrial process. In addition, recombinant OepHPL could be identified as the HPL activity in crude preparations. The thermal stability was the highest among the recombinant proteins studied, which suggests that HPL activity is not a limiting factor for the synthesis of VOO volatile compounds.

The Entner-Doudoroff pathway is obligatory for gluconate utilization and contributes to the pathogenicity of Vibrio cholerae.

The Entner-Doudoroff (ED) pathway has recently been shown to play an important role in sugar catabolism for many organisms although very little information is available on the functionality of this pathway in Vibrio cholerae, the causative agent of cholera. In this study, activation of the genes edd and eda, encoding 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase, was used as a marker of a functional ED pathway in V. cholerae. Transcriptional activation analyses and gene silencing experiments with cells grown in sugar-supplemented M9 medium demonstrated that the ED pathway is functional in V. cholerae and is obligatory for gluconate catabolism. Importantly, selective activation of the ED pathway led to concurrent elevation of transcripts of prime virulence genes (ctxA and tcpA) and their regulator (toxT). Further, lowering of these transcript levels and cholera toxin production in vitro by an ED pathway-defective mutant (strain N16961 with a Δedd mutation [Δedd(N16961) strain]) suggested the importance of this pathway in regulating V. cholerae virulence. The in vivo relevance of these data was established as the mutant failed to colonize in suckling mice intestine or to induce fluid accumulation in ligated rabbit ileal loops. Activation of the ED pathway in V. cholerae was shown to inhibit biofilm formation in vitro that could be reversed in the mutant. As further support for these results, comparative transcriptome analysis with cells grown in the presence of glucose or gluconate revealed that a functional ED pathway led to activation of a subset of previously reported in vivo expressed genes. All of these results suggest the importance of the ED pathway in V. cholerae pathogenesis.

Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

The D-galacturonic acid catabolic pathway in Botrytis cinerea.

D-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving D-galacturonate reductase, L-galactonate dehydratase, and 2-keto-3-deoxy-L-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire D-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the D-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.