Expression and Secretion of a Lytic Polysaccharide Monooxygenase by a Fast-growing Cyanobacterium

Background: Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO, light and inorganic nutrients to produce a range of biomolecules of commercial interest. UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant AA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium

Results: Two variations of AA10A were successfully expressed in UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the trimethylamine--oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-AA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of AA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. AA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.

Conclusions: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.