Enhanced Scytonemin Biosynthesis in Engineered Nostoc flagelliforme with ScyR Overexpression and Cultivation Optimization

Scytonemin, a lipid-soluble indole-alkaloid pigment unique to cyanobacteria, is a bioactive small molecule with UV-shielding, antioxidant, and anti-inflammatory activities. Nostoc flagelliforme represents a promising strain for scytonemin biosynthesis, yet engineering to boost its yield remains challenging. Two genes encoding a histidine kinase (COO91_00772) and a response regulator (COO91_00771, ScyR) putatively constitute a two-component regulatory system governing the UV-induced biosynthesis of scytonemin. Here, we employed CRISPR/Cpf1 technology to achieve the insertion of an additional ScyR expression cassette into the N. flagelliforme genome and evaluated its effect on scytonemin yield. The engineered strain was designated Nf-psbA-ScyR, in which the ScyR gene was expressed under the PpsbA promoter. This strain exhibited a markedly enhanced accumulation of scytonemin, reaching 4.6 mg g−1 dry weight under UV-B radiation after 15-day cultivation with exogenous tryptophan supplementation, representing a 1.9-fold increase compared with the MAAs-deficient (Nf-MAAs-D) strain. Upon transition from indoor UV-B exposure to outdoor solar radiation, scytonemin production further increased to 5.4 mg g−1 dry weight. These findings indicate that the ScyR regulator can highly promote scytonemin biosynthesis under UV induction, providing an engineering strategy for the exploitation of N. flagelliforme.