Optimizing photoautotrophic production of soluble 3-hydroxybutyrate in Synechocystis sp. PCC 6803 through combinatorial translational tuning

While photosynthetic cyanobacteria are potential biotechnological hosts for light-driven production of sustainable chemicals from CO2, engineering more efficient strains is critical for the development of competitive industrial processes. This study demonstrates significantly enhanced production of the soluble bioplastic precursor (R)-3-hydroxybutyrate (3HB) that has been engineered based on the polyhydroxybutyrate (PHB) pathway in photoautotrophic cyanobacterium Synechocystis sp PCC 6803. As the key novelty, we generated a library of engineered 3HB pathway variants that express the three key heterologous pathway enzymes PhaA, PhaB and TesB at varying efficiencies, followed by the screening of most efficient 3HB producers. This was achieved by placing each of the pathway enzymes under the translational regulation of three alternative RBSs in different combinations, resulting in strains with wide dynamic range of 3HB productivities. The best strains accumulated over 5 gl-1 under 200 mol photons m-2s-1 and 3% CO2 in a 14-day flask batch culture, with the highest titer reaching 12 gl-1, corresponding to nearly 3 gl-1 d-1 during the peak production phase. These are the highest 3HB production levels reported so far in cyanobacteria, and comparable to those previously established in heterotrophic production systems. Proteomic comparison of selected strains revealed that the different RBS combinations result in varying expression patterns of the pathway proteins, and that the strain-specific enzyme levels remained relatively constant over the monitored six-day period. The results show that altering the levels of the target pathway enzymes can dramatically improve product yield in Synechocystis, while even very small quantitative differences in the strain-specific expression profiles can have marked effects on the production efficiency. This could be a general tool for optimizing engineered pathways in cyanobacteria, provided that the flux to the end-product is not critically restricted by substrate availability but determined by the balance between the consecutive pathway steps.