Examinando por Autor "Wittmann, Christoph"

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    Cascaded valorization of brown seaweed to produce L-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering
    (Academic Press Inc., 2021-09) Hoffmann, Sarah Lisa; Kohlstedt, Michael; Jungmann, Lukas; Hutter, Michael; Poblete-Castro, Ignacio; Becker, Judith; Wittmann, Christoph
    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.
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    Ítem
    Production of medium chain length polyhydroxyalkanoate in metabolic flux optimized Pseudomonas putida
    (BioMed Central Ltd., 2014-06) Borrero-de Acuña, José M.; Bielecka, Agata; Häussler, Susanne; Schobert, Max; Jahn, Martina; Wittmann, Christoph; Jahn, Dieter; Poblete-Castro, Ignacio
    Background: Pseudomnas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A two-fold increase of mcl-PHA production via inactivation of the glucose dehydrogenase gene gcd, limiting the metabolic flux towards side products like gluconate was achieved before. Here, we investigated the overproduction of enzymes catalyzing limiting steps of mcl-PHA precursor formation. Results: A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type and the Δgcd mutant strains led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type and 121% in the acoA overexpressing Δgcd strain in comparison to P. putida KT2440. Overexpression of pgl-encoding 6-phosphoglucolactonase did not influence PHA production. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms. Conclusion: Production of mcl-PHAs was enhanced in P. putida when grown on glucose via overproduction of a pyruvate dehydrogenase subunit (AcoA) in combination with a deletion of the glucose dehydrogenase (gcd) gene as predicted by in silico elementary flux mode analysis.