Abstract
D-Mannitol production was achieved in freshwater Synechocystis sp. PCC6803 via the heterologous expression of mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p) under control of the strong promoter P(trc1). However, only 5.54 mg L(-1) of mannitol was found extracellularly after 7 days of cultivation, likely due to insufficient expression of a mutated mtlD lacking a methionine at position 332. This study compared mannitol levels using different promoters (P(trc1), P(psbA2) and P(nrsB)) to control the expression of (un)mutated versions of mtlD in Synechocystis with co-expression of m1p. Our data suggest that even without the inducer, the weakest promoter, P(nrsB), can support the expression of an unmutated mtlD in Synechocystis, which leads to 18.2 mg L(-1) of mannitol in 7 days without induction. Such titer is already much higher than the first engineered mannitol-producing Synechocystis. When 5 μM nickel sulfate was added to the medium as an inducer, mannitol production could significantly be increased further, up to 92.9 mg L(-1) after 7 days of induction, but it partially inhibited growth. Attempts with the other increasingly stronger promoters always failed to express the unmutated mtlD, probably due to the toxicity caused by the accumulation of the intermediate product, mannitol-1-phosphate. These results clearly suggest that the expression level of mtlD is the bottleneck in achieving a high yield of mannitol in Synechocystis, and consequently, that mannitol production can be enhanced by fine-tuning its expression. Future research is needed to identify bottlenecks that hinder mannitol productivity and long-term stability, facilitating the engineering of more efficient mannitol-producing cyanobacterial strains.