Effects of Overexpressing Calvin-Bassham-Benson Cycle Genes in Synechocystis PCC 6803

Liang, F., Uppsala University
Lindblad, P., Uppsala University
It is environmentally sustainable to convert inorganic carbon directly into desired compounds using photoautotrophic organisms, like higher plants, algae, and cyanobacteria. The Calvin-Bassham-Benson (CBB) cycle is the primary carbon fixation pathway in photoautotrophic organisms. Synechocystis PCC 6803 (Synechocystis) is a unicellular model cyanobacterium used in e.g. photosynthesis and CO2 assimilation research. In the present study, we examined the effects of overexpressing Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7- biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the CBB cycle in higher plants, in Synechocystis.

Two different strategies were used to increase the RuBisCO content in the cells. One was to introduce ccmMgene into wild type cells on a self-replication vector (Liang and Lindblad, 2016) and the second was to add a FLAG tag sequence on the large subunit of the N terminus or the small subunit of the C terminus of RuBisCO. Since CcmM is a carboxysome protein that binds RuBisCO, CcmM may localize RuBisCO into the carboxysome as well as stabilize the enzyme. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation and/or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe similar effects. FBP/SBPase (di-functional enzyme in cyanobacteria), FBA and TK were successfully overexpressed by introducing another gene copy on self-replication vector (Liang and Lindblad, 2016).

The results demonstrate that overexpressing above discussed enzymes in Synechocystis resulted in increased in vivo oxygen evolution. Under 100μmol m-2 s-1 irradiance, growth and biomass were enhanced in cells overexpressing RuBisCO, FBP/SBPase and FBA. Cells overexpressing TK behaved differently, showing slower growth but increased biomass accumulation. In addition, 20μmol m-2 s-1 irradiance restricted the engineered strains' growth and biomass accumulation. However, cells overexpressing TK showed increased biomass under 20μmol m-2 s-1 irradiance. This study demonstrates enhanced growth and photosynthesis in cells of the cyanobacterium Synechocystis overexpressing selected enzymes of the CBB cycle. RuBisCO, SBP/FBPase, FBA and TK are four potential targets to improve growth of, and yield of valuable products from, SynechocystisPCC 6803.

Feiyan Liang, Peter Lindblad*, Effects of Overexpressing Calvin-Bassham-Benson Cycle Carbon Flux Control Enzymes in the Cyanobacterium Synechocystis PCC 6803. (2016) Metabolic Engineering 38:56-64. doi: 10.1016/j.ymben.2016.06.005.