Augmenting the Calvin-Benson-Bassham cycle by a synthetic malyl-CoA-glycerate carbon fixation pathway.

The Calvin-Benson-Bassham (CBB) cycle is presumably evolved for optimal synthesis of C3 sugars, but not for the production of C2 metabolite acetyl-CoA. The carbon loss in producing acetyl-CoA from decarboxylation of C3 sugar limits the maximum carbon yield of photosynthesis. Here we design a synthetic malyl-CoA-glycerate (MCG) pathway to augment the CBB cycle for efficient acetyl-CoA synthesis. This pathway converts a C3 metabolite to two acetyl-CoA by fixation of one additional CO2 equivalent, or assimilates glyoxylate, a photorespiration intermediate, to produce acetyl-CoA without net carbon loss. We first functionally demonstrate the design of the MCG pathway in vitro and in Escherichia coli. We then implement the pathway in a photosynthetic organism Synechococcus elongates PCC7942, and show that it increases the intracellular acetyl-CoA pool and enhances bicarbonate assimilation by roughly 2-fold. This work provides a strategy to improve carbon fixation efficiency in photosynthetic organisms.

Metabolic engineering of cyanobacteria for photosynthetic 3-hydroxypropionic acid production from CO2 using Synechococcus elongatus PCC 7942.

Photosynthetic conversion of CO2 to chemicals using cyanobacteria is an attractive approach for direct recycling of CO2 to useful products. 3-Hydroxypropionic acid (3 HP) is a valuable chemical for the synthesis of polymers and serves as a precursor to many other chemicals such as acrylic acid. 3 HP is naturally produced through glycerol metabolism. However, cyanobacteria do not possess pathways for synthesizing glycerol and converting glycerol to 3 HP. Furthermore, the latter pathway requires coenzyme B12, or an oxygen sensitive, coenzyme B12-independent enzyme. These characteristics present major challenges for production of 3 HP using cyanobacteria. To overcome such difficulties, we constructed two alternative pathways in Synechococcus elongatus PCC 7942: a malonyl-CoA dependent pathway and a ß-alanine dependent pathway. Expression of the malonyl-CoA dependent pathway genes (malonyl-CoA reductase and malonate semialdehyde reductase) enabled S. elongatus to synthesize 3 HP to a final titer of 665 mg/L. ß-Alanine dependent pathway expressing S. elongatus produced 3H P to final titer of 186 mg/L. These results demonstrated the feasibility of converting CO2 into 3 HP using cyanobacteria.