Employing Nanotechnology to Enhance Ethanol Production in Two Synechococcus elongatus Strains

Cyanobacteria have emerged as promising photosynthetic cell factories. However, low product yields remain a bottleneck to industrial exploitation. Here, we use bacterial microcompartment technology to improve product titres using ethanol biosynthesis as the model pathway. Two strategies compared to unmodified enzyme production were tested: first, targeting enzymes to nanofilaments by fusion with encapsulation peptides (EPs) which form non-covalent interactions with the filaments. Second, the enzyme-EP fusions were used in the absence of nanofilaments, as EPs tend to form soluble aggregates. Both approaches were tested in Synechococcus elongatus PCC 7942 and the fast-growing Synechococcus elongatus UTEX 2973 to validate if fast growth is additionally beneficial for bioproduction. The EPs had the most dramatic effect on enzyme levels and ethanol production leading to an increase in titres of up to 84 times in comparison to the unmodified enzymes. While Se7942 exhibited higher titres per cell, titres were the highest in Se2973 due to a 79% increase in final cell density. Ultimately, the best strategy (enzyme-EP fusion in the absence of PduA*) led to 1.31 g L^-1 of ethanol in 6 days. This work demonstrates that nanotechnology-based strategies can aid cyanobacterial bioproduction and will serve as a blueprint for future engineering efforts.