Heterologous expression and functional characterization of two alginate lyases in corynebacterium glutamicum

Alginates are linear, anionic polysaccharides consisting of β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G), organized in block structures of M, G, and alternating MG-blocks. Alginates are abundant in brown seaweeds, and seaweed extracts have a large potential as sustainable feedstock for industrial biotechnology. Here, the alyB and alyD genes associated with alginate degradation in Vibrio algivorus are heterologously expressed and secreted by Corynebacterium glutamicum. The mode of action of AlyB and AlyD, individually or in combination, were characterized using NMR spectroscopy, HPAEC-PAD, and an absorbance-based assay. AlyB was found to be endo-active degrading all types of alginates (G blocks, M block, GM) bonds forming short oligomers. AlyB also displayed C-5 epimerization activity. AlyD on the other hand, is exo-active when using the products formed by AlyB depolymerization as substrates, thus AlyD degrades Δ-containing oligomers into Δ monosaccharides. However, AlyD is not active on saturated oligomers. Surprisingly, when acting together, AlyB and AlyD synergistically depolymerize alginates to Δ monosaccharides. The heterologously expressed and secreted AlyB and AlyD in C. glutamicum enabled the breakdown of alginate to the uronate monomers β-D-mannuronate and α-L-guluronate. The resulting substrate was here used as feedstock for growth of Escherichia coli K12 MG1655. Moreover, by using a genetically engineered E. coli MG1655 strain expressing a synthetic riboflavin operon we demonstrated riboflavin production to a concentration of 2.1 ± 0.1 µg/mL using alginate depolymerized by AlyB and AlyD heterologously produced in C. glutamicum as sole carbon source. Engineered C. glutamicum secretes active alginate lyases from V. algivorus. Biochemical data shows distinct action modes of the alginate lyases AlyB and AlyD. AlyB and AlyD act synergistically to enhance alginate depolymerization. AlyB and AlyD generate defined uronate monomers from alginate. Engineered E. coli produces riboflavin from alginate-derived uronates.