Genetic engineering of Synechocystis sp PCC6803 for poly-β-hydroxybutyrate overproduction

The biosynthesis of poly-beta-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that (i) the PHB synthesis pathway becomes deregulated, and/or (ii) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve. The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232 mg L-1, similar to 12% (w/w) of the dry biomass weight, and a productivity of 7.3 mg L-1 d(-1); are to the best of our knowledge, the highest ever reported for PHB production directly from CO2.