Efficient selection of pyruvate decarboxylase sequences from database for high ethanol productivity in Synechocystis sp. PCC 6803.

Ethanol production using the model cyanobacterium Synechocystis sp. PCC 6803 (PCC6803) has garnered considerable attention. A heterologous pyruvate decarboxylase (PDC) is essential for synthesizing ethanol in PCC6803. Although many organisms possess PDCs, no systematic search for suitable PDCs has been reported. This study employed a two-step approach to identify promising PDCs. First, nine diverse natural PDCs with confirmed activity in BRENDA were evaluated for ethanol production in PCC6803. Ethanol production was observed only with PDCs from Zymomonas mobilis (Zm PDC) and Gluconobacter diazotrophicus, suggesting that bacterial PDCs are suitable. In the second step, the search focused on bacterial PDCs, not only natural PDCs but also artificial sequences designed via the Protein Repair One-Stop Shop or ancestral sequence reconstruction. A PDC from Gluconobacter oxydans showed higher ethanol productivity (88.9 mg/L/5 days) than Zm PDC. Although productivity did not surpass that of Zm PDC, ethanol production was achieved with previously unconfirmed or engineered PDCs, expanding the range of useable sequences. This stepwise strategy demonstrates a robust approach for identifying and designing useful enzymes across sequence spaces.