Enhanced sucrose production by controlling carbon flux through CfrA expression in Synechocystis sp. PCC 6803

Abstract

BACKGROUND: Cyanobacteria, as phototrophic organisms with low nutritional requirements and great metabolic versatility, are attractive for the sustainable production of value-added chemicals from CO2 and sunlight. One limitation of these strategies is that carbon is partitioned towards biomass synthesis rather than product synthesis. An alternative to conventional metabolic engineering approaches involves controlling regulatory circuits to enhance the flow of carbon towards the synthesis of desired compounds. The carbon-flow-regulator A (CfrA) is pivotal in redirecting carbon flux during nitrogen deficiency in cyanobacteria, promoting glycogen accumulation by inhibiting 2,3-phosphoglycerate mutase enzyme. The moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803 accumulates sucrose and glucosylglycerol (GG) as compatible solutes under salt stress. Sucrose is a valuable carbon source for heterotrophic organisms, whether they are cultivated independently or in co-cultures. In this context, we explored the potential biotechnological relevance of CfrA in redirecting carbon flow towards sucrose production. RESULTS: A strain that overexpresses cfrA, independently of nitrogen growth conditions, and carries a plasmid that expresses sucrose-phosphate synthase (SPS) from Synechocystis sp. PCC 6803 and the heterologous sucrose permease CscB inducibly (Pars-cfrA/suc strain) was constructed and analysed. In this strain, cfrA expression increased sucrose production by 40% compared to non-induced levels. The fixed carbon was partially redirected towards sucrose production at the expense of glycogen accumulation and biomass generation. Furthermore, an improvement in the photosynthetic activity of this strain was observed due to the presence of this carbon sink. The effect of eliminating GG synthesis (ΔggpS/Pars-cfrA/suc strain) on sucrose production was also analyzed. Under high salinity conditions (400 mM NaCl), this strain exhibited a maximum sucrose accumulation of 2.72 g/L. Encapsulation of the Pars-cfrA/suc strain has also been studied. CONCLUSIONS: Our results indicate that modulating carbon flow through CfrA overexpression can substantially boost sucrose production. Glycogen accumulation, mediated by CfrA, enhances sucrose production, which is partly derived from the use of stored glycogen. Furthermore, immobilising Synechocystis cells in alginate improves sucrose production and facilitates its utilisation. Given the widespread occurrence of the cfrA gene in cyanobacteria, its potential as a target in various biotechnological strategies that require the redirection of carbon flow should be considered.

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