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

Abstract 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.