Expression landscape of metabolic engineering enzymes in cyanobacteria

Abstract

Photosynthetic cyanobacteria are promising platforms for sustainable chemical production, as they can convert light and CO2 into valuable compounds. Achieving this often requires engineering cyanobacteria with non-native enzymes with strong promoters to maximize enzyme accumulation. However, despite extensive engineering efforts, the extent to which these enzymes misfold and undergo degradation in cyanobacteria remains unknown. Here, we systematically investigate the fate of recombinant proteins in Synechocystis sp. PCC 6803 by estimating protein loss due to protease degradation. To do this, we developed a quantitative approach that combines split-GFP reporting with inducible CRISPRi knockdown of Clp protease system, enabling estimation of portion of proteins that would otherwise be degraded. Applying this method to 103 heterologous proteins previously used in cyanobacterial metabolic engineering studies, we find that, on average, one-third of recombinant protein accumulation is lost to degradation, with some enzymes exhibiting more than 95% protein loss. Furthermore, we compare expression from identical expression constructs in E. coli and Synechocystis and find broad similarities in their protein accumulation patterns. Together, these findings provide the first quantitative overview of heterologous protein expression in cyanobacteria and identify enzymes that are suboptimal for their respective pathways, information usable to increase production titers in photosynthetic cell factories.