Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium |
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| Institution: | 1. Photanol BV, Science Park 408, 1098 XH Amsterdam, the Netherlands;2. Swammerdam Institute for Life Sciences, University of Amsterdam, the Netherlands;1. Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, MO 63130, USA;2. Donald Danforth Plant Science Center, St. Louis, MO 63132, USA;3. United States Department of Agriculture, Agricultural Research Service, St. Louis, MO 63132, USA;4. Department of Biochemistry, University of Colorado Boulder, Boulder, CO 80309, USA;5. Renewable and Sustainable Energy Institute, University of Colorado, Boulder, CO 80309, USA;6. National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401, USA;1. Biosciences Center, National Renewable Energy Laboratory, Golden, CO, USA;2. Institute of Plant Biology, National Taiwan University, Taipei, Taiwan;3. Institute of Pharmacology, Kaohsiung Medical University, Kaohsiung, Taiwan;4. Department of Genome Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan |
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| Abstract: | d-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L?1 and a production rate of 0.15 g mannitol L?1 day?1. This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria. |
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| Keywords: | Mannitol Cyanobacteria Synthetic biology Bio-based chemicals Rare sugars |
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