Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L⁻¹ l-lysine from mannitol at a yield of 0.26 mol mol⁻¹ and a maximum productivity of 2.1 g L⁻¹ h⁻¹. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol⁻¹ using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol⁻¹. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.     

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

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How the insect immune system interacts with an obligate symbiotic bacterium

The animal immune system provides defence against microbial infection, and the evolution of certain animal–microbial symbioses is predicted to involve adaptive changes in the host immune system to accommodate the microbial partner. For example, the reduced humoral immune system in the pea aphid Acyrthosiphon pisum, including an apparently non-functional immune deficiency (IMD) signalling pathway and absence of peptidoglycan recognition proteins (PGRPs), has been suggested to be an adaptation for the symbiosis with the bacterium Buchnera aphidicola. To investigate this hypothesis, the interaction between Buchnera and non-host cells, specifically cultured Drosophila S2 cells, was investigated. Microarray analysis of the gene expression pattern in S2 cells indicated that Buchnera triggered an immune response, including upregulated expression of genes for antimicrobial peptides via the IMD pathway with the PGRP-LC as receptor. Buchnera cells were readily taken up by S2 cells, but were subsequently eliminated over 1–2 days. These data suggest that Buchnera induces in non-host cells a defensive immune response that is deficient in its host. They support the proposed contribution of the Buchnera symbiosis to the evolution of the apparently reduced immune function in the aphid host.     

Metabolic effects of anti-angiogenic therapy in tumors

Anti-angiogenic therapy has recently been added to the panel of cancer therapeutics, but predictive biomarkers of response are still not available. In animal models, anti-angiogenic therapy causes tumor starvation by increasing hypoxia and impairing nutrients supply. It is thus conceivable that angiogenesis inhibition causes remarkable metabolic perturbations in tumors, although they remain largely uncharted. We review here recent acquisitions about metabolic effects of angiogenesis blockade in tumors and discuss the possibility that some metabolic features of tumor cells - i.e. their dependency from glucose as primary energy substrate - might affect tumor responses to anti-VEGF treatment.     

Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment.     

Demethylation and degradation of phenylmethylethers by the sulfide-methylating homoacetogenic bacterium strain TMBS 4

Biochemical studies on anaerobic phenylme-thylether cleavage by homoacetogenic bacteria have been hampered so far by the complexity of the reaction chain involving methyl transfer to acetyl-CoA synthase and subsequent methyl group carbonylation to acetyl-CoA. Strain TMBS 4 differs from other demethylating homoacetogenic bacteria in using sulfide as a methyl acceptor, thereby forming methanethiol and dimethylsulfide. Growing and resting cells of strain TMBS 4 used alternatitively CO₂ as a precursor of the methyl acceptor CO for homoacetogenic acetate formation. Demethylation was inhibited by propyl iodide and reactivated by light, indicating involvement of a corrinoid-dependent methyltransferase. Strain TMBS 4 contained ca. 750 nmol g dry mass^-1 of a corrinoid tentatively identified as 5-hydroxybenzimidazolyl cobamide. A photometric assay for measuring the demethylation activity in cell extracts was developed based on the formation of a yellow complex of Ti³⁺ with 5-hydroxyvanillate produced from syringate by demethylation. In cell extracts, the methyltransfer reaction from methoxylated aromatic compounds to sulfide or methanethiol depended on reductive activation by Ti³⁺. ATP and Mg²⁺ together greatly stimulated this reductive activation without being necessary for the demethylation reaction itself. The specific activity of the transmethylating enzyme system increased proportionally with protein concentration up to 3 mg ml^-1 reaching a constant level of 20 nmol min^-1 mg^-1 at protein concentrations 10 mg ml^-1. The specific rate of activation increased in a non-linear manner with protein concentration. Strain TMBS 4 degraded gallate, the product of sequential demethylations, to 3 acetate through the phloroglucinol pathway as found earlier with Pelobacter acidigallici.Abbreviations BV benzyl viologen - CTAB cetyltrimethylammonium bromide - H₄folate tetrahydrofolate - MOPS 3-[N-morpholino]propanesulfonic acid - MV methyl viologen - NTA nitrilotriacetate - t_d doubling time - TMB 3,4,5-trimethoxybenzoate     

Notch signaling: a mediator of beta-cell de-differentiation in diabetes?

Cytokines are mediators of pancreatic beta-cell dysfunction and death in type 1 diabetes mellitus. Microarray analyses of insulin-producing cells exposed to interleukin-1beta+interferon-gamma showed decreased expression of genes related to beta-cell-differentiated functions and increased expression of members of the Notch signaling pathway. Re-expression of this developmental pathway may contribute for loss-of-function of beta-cells exposed to an autoimmune attack. In this study, we show that rat primary beta-cells exposed to cytokines up-regulate several Notch receptors and ligands, and the target gene Hes1. Transfection of insulin-producing INS-1E cells and primary rat beta-cells with a constitutively active form of the Notch receptor down-regulated Pdx1 and insulin expression in INS-1E cells but not in primary beta-cells. Thus, activation of the Notch pathway inhibits differentiated functions in dividing but not in terminally differentiated beta-cells.     

Characterization of Tiki,a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn²⁺/Co²⁺ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000