Research on biofuel production by cyanobacteria has largely focused on unicellular species. To date, there are no reports on engineered filamentous cyanobacteria for bioethanol or biohydrogen production. However, biofilm formation in filamentous cyanobacteria could provide an advantage for biotechnological applications. We used natural transformation and homologous recombination to establish recombinant expression at the psaD locus of the filamentous cyanobacterium Phormidium lacuna. A similar approach has already been successful in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Appel et al. 2020). Fusion of the NiFe hydrogenase subunits HoxY/HoxH from Microcystis aeruginosa to the PSI subunit PsaD increased hydrogen production five-fold under light in the presence of DCMU, although dark fermentative hydrogen levels remained higher. Structural modeling indicates that electron transfer may be limited by the FeS cluster distance, suggesting opportunities for further optimization. Using sfGFP fluorescence, we showed that the psaD promoter drives strong expression when the gene is positioned 3′ of the psaD gene or expressed as a PsaD–sfGFP fusion protein. The psaD promoter was then used for expression of pyruvate decarboxylase and alcohol dehydrogenase, resulting in ethanol production of 0.2‰ (v/v) and outperforming the kanamycin promoter used in previous unsuccessful trials. These results demonstrate that P. lacuna is a robust host for biohydrogen and bioethanol production and validate the psaD promoter as a strong regulatory element for filamentous cyanobacteria in biotechnological applications.
