1. Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering
- Author
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Sarah Lisa Hoffmann, Ignacio Poblete-Castro, Michael Kohlstedt, Lukas Jungmann, Judith Becker, Christoph Wittmann, and Michael C. Hutter
- Subjects
Lysine ,Bioengineering ,Fructose ,Oxidative pentose phosphate pathway ,Applied Microbiology and Biotechnology ,Corynebacterium glutamicum ,Metabolic engineering ,Transhydrogenase ,chemistry.chemical_compound ,Redox balancing ,Fructokinase ,Arabitol ,Glyceraldehyde 3-phosphate dehydrogenase ,NADPH ,Humans ,Food science ,Mannitol 2-Dehydrogenase ,L-lysine ,Macro algae ,Seaweed ,Bioproduction ,Metabolic Engineering ,Mannitol dehydrogenase ,chemistry ,Mannitol 2-dehydrogenase ,NADH ,bacteria ,Fermentation ,Protein engineering ,NADP ,Biotechnology - Abstract
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.
- Published
- 2021