Photosynthetic sorbitol production in Synechococcus sp. PCC 7002 is enhanced by addressing phosphatase promiscuity, nutrient availability and Calvin cycle bottlenecks.

Cyanobacteria represent promising biocatalysts for producing carbohydrates, including sorbitol, a naturally-occurring, fermentable sugar alcohol with conventional uses as a sweetener, pharmaceutical additive, and biodegradable plasticizer. Previously, Synechocystis sp. PCC 6803 was engineered to produce sorbitol, reaching a final titer of 2.3 g/L after 18 days. To improve upon this performance, sorbitol production was herein engineered in the faster growing strain Synechococcus sp. PCC 7002. Upon introducing the sorbitol biosynthetic pathway, up to 500 mg/L sorbitol was initially produced after seven days. However, due to the initial use of two highly promiscuous sugar phosphatase variants, this also resulted in the unwanted co-production of ribose and growth inhibition due to depletion of ribose-5-phosphate from the Calvin cycle. This off-target effect was ultimately mitigated via the discovery that mannitol-1-phosphate phosphatase from Eimeria tenella also dephosphorylates sorbitol-6-phosphate to sorbitol with greater specificity, leading to improved growth and sorbitol production. Next, two bottleneck enzymes in the Calvin cycle, namely fructose-bisphosphate aldolase (FBA) and bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase (BiBPase), were overexpressed both individually and in combination, resulting in sorbitol production up to 1.3 g/L. Finally, upon optimizing the culture media to address nutrient limitation, the final strain produced up to 3.6 g/L sorbitol in nine days, respectively representing 1.5- and 3-fold increases in titer and productivity relative to previously-engineered Synechocystis sp. PCC 6803.