Abstract
Fructose is an important monosaccharide product widely applied in the food, medicine, and chemical industries. Currently, fructose is mainly manufactured with plant biomass-sourced polysaccharides through multiple steps of digestion, conversion, separation, and purification. The development of cyanobacterial metabolic engineering provides an attractive alternative route for the one-step direct production of fructose utilizing carbon dioxide and solar energy. In this work, we developed a paradigm for engineering cyanobacterial chassis cells into efficient cell factories for the photosynthetic production of fructose. In a representative cyanobacterial strain, Synechococcus elongatus PCC 7942, knockout of fructokinase effectively activated the synthesis and secretion of fructose in hypersaline conditions, independent of any heterologous transporters. The native sucrose synthesis pathway was identified as playing a primary role in fructose synthesis. Through combinatory optimizations on the levels of metabolism, physiology, and cultivation, the fructose yield of the Synechococcus cell factories was stepwise improved to 3.9 g/L. Such a paradigm was also adopted to engineer another Synechococcus strain, the marine species Synechococcus sp. PCC 7002, and facilitated an even higher fructose yield of over 6 g/L. Finally, the fructose synthesized and secreted by the cyanobacterial photosynthetic cell factories was successfully extracted and prepared from the culture broth in the form of products with 86% purity through multistep separation-purification operations. This work demonstrated a paradigm for systematically engineering cyanobacteria for photosynthetic production of desired metabolites, and it also confirmed the feasibility and potential of cyanobacterial photosynthetic biomanufacturing as a simple and efficient route for fructose production.
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