Introduction of acetyl-phosphate bypass and increased culture temperatures enhanced growth-coupled poly-hydroxybutyrate production in the marine cyanobacterium Synechococcus sp. PCC7002.

Polyhydroxyalkanoate (PHA) is an attractive bio-degradable plastic alternative to petrochemical plastics. Photosynthetic cyanobacteria accumulate biomass by fixing atmospheric CO, making them promising hosts for sustainable PHA production. Conventional PHA production in cyanobacteria requires prolonged cultivation under nutrient limitation to accumulate cellular PHA. In this study, we developed a system for growth-coupled production of the PHA poly-hydroxybutyrate (PHB), using the marine cyanobacterium Synechococcus sp. PCC 7002. A recombinant strain termed KB1 expressing a set of heterologous PHB biosynthesis genes (phaA/phaB from Cupriavidus necator H16 and phaE/phaC from Synechocystis sp. PCC 6803) accumulated substantial PHB during growth (11.4% of dry cell weight). To improve PHB accumulation, we introduced the Pseudomonas aeruginosa phosphoketolase gene (pk) into strain KB1, rewiring intermediates of the Calvin-Benson-Bassham (CBB) cycle (xyluose-5-phosphate, sedoheptulose 7-phosphate, and fructose-6-phosphate) to acetyl-CoA. The pk-expressing strain, KB15, accumulated 2.1-fold enhanced levels of PHB (23.8% of dried cell weight), relative to the parent strain, KB1. The highest PHB titer of KB15 strain supplemented with acetate was about 1.1 g L and the yield was further enhanced by 2.6-fold following growth at 38°C (0.21 g L d), relative to growth at 30°C. Metabolome analysis revealed that pool sizes of CBB intermediates decreased, while levels of acetyl-CoA increased in strain KB15 compared with strain KB1, and this increase was further enhanced following growth at 38°C. Our data demonstrate that acetyl-phosphate generated by Pk was converted into acetyl-CoA via acetate by hitherto unidentified enzymes. In conclusion, expression of heterologous PHB biosynthesis genes enabled growth-coupled PHB production in strain PCC 7002, which was increased through acetyl-CoA supplementation by bypassing acetyl-phosphate and elevating culture temperature.