(614a) Photosynthetic Sorbitol Production By Engineered Synechococcus Sp. PCC 7002 and Its Impacts on CO2 Fixation

Cyanobacteria represent attractive biocatalysts due to their ability to photosynthetically fix CO₂ and convert it into biomass and value-added chemicals. Cyanobacteria naturally synthesize sugars such as sucrose, fructose and glucose via the Calvin cycle, and strains capable of overproducing sucrose, mannitol, erythritol and sorbitol have all previously been engineered. Sorbitol, in particular, is used as food additive as a low-calorie sugar substitute, food stabilizer or emulsifier, and can also serve as a precursor to propylene glycol, sorbose and vitamin C. Bacterial sorbitol production begins via the NADPH-dependent reduction of glucose-6-phosphate (G6P) to sorbitol-6-phosphate, which can then be further converted to sorbitol by a polyol phosphatase enzyme (both yeast and E. coli variants were evaluated herein). Previous efforts to engineer sorbitol production in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803) achieved a titer of ~400 mg/L sorbitol after seven days. However, since relative to PCC 6803, Synechococcus sp. PCC 7002 (PCC 7002) displays a faster growth rate, higher biomass accumulation and better tolerance to higher temperatures and salinity, here we elected to explore the engineering of PCC 7002 for direct photosynthetic production of sorbitol. First, by simply introducing the sorbitol biosynthetic pathway without further strain optimization, PCC 7002 could produce 500 mg/L sorbitol after 7 days, a 25% improvement over PCC 6803. Furthermore, we have tested a recently discovered yeast polyol phosphatase gene with sorbitol-6-phosphatase activity and found that sorbitol production using this enzyme is indistinguishable to the previously discovered E. coli polyolphosphatase. Expression of the yeast polyol phosphatase enzyme also notably led to the co-production of glycerol, indicating that it also displays glycerol-6-phosphate phosphatase activity. Further improvements of sorbitol production are now also being investigated via the expression of a sorbitol transporter. Several sugar alcohol-H⁺ symporters are being individually cloned and introduced into our PCC 7002 strains to screen for their ability to enhance the extracellular accumulation and overall production of sorbitol.

Next, to fix and ‘push’ additional carbon toward sorbitol production, additional copies of key Calvin cycle genes have been integrated into and expressed from the genome of PCC 7002 to both individually and combinatorially test for synergistic improvements to sorbitol production. Specifically, four Calvin cycle genes are typically considered as potential bottlenecks: transketolase (TK), bifunctional fructose-1,6-/seduheptoluse-1,7-biphosphatase (FBP/SBPase), Fructose-bisphosphate aldolase (FBA), and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Of these, three – TK, FBA, and FBP/SBPase – are responsible for regeneration of ribulose bisphosphate (RuBP). Among these, it is hypothesized that co-expression of FBA and FBP/SBPase will be of particular interest since their direct product – fructose-6-phosphate (F6P) – is a direct precursor to sorbitol production.

Finally, the impacts of sorbitol production on the rates and level of carbon fixation by PCC 7002 are being assessed via high-resolution CO₂ uptake studies using a novel, automated off-gas analyzer system. Using this system, we have demonstrated that when wild type PCC 7002 is grown at 37°C, 1% CO₂ and 300 µmol photons m^-2 s^-1 the CO₂ fixation rate follows a sigmoidal curve with peak fixation occurring between 24 and 48 hours. It was previously shown for the case of sucrose overproduction that, relative to the wild type, carbon fixation, biomass accumulation and photosynthetic efficiency were all increased, suggesting that photosynthesis was constrained by absence of an amenable, non-biomass carbon/electron sink. Interestingly, for the PCC 7002 strains constructed here, sorbitol production appears to continue for multiple days after the cells enter stationary phase, suggesting that photosynthetic carbon fixation continues in non-growing cultures. Here, we quantitatively assess the benefits and limits of these observed behaviors to maximize CO₂ removal and sorbitol production.