Using Osmotic Stress to Stabilize Mannitol Production in Synechocystis sp. PCC6803

Abstract

<jats:title>Abstract</jats:title> <jats:p>Background Mannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO 2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC 7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase ( mtlD ) and mannitol-1-phosphatase ( m1p , in short: a ‘mannitol cassette’), proved to be genetically unstable. Results Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC 6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC 6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild type Synechocystis , complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production. Conclusions Mannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of both stable mannitol production directly from CO 2 in cyanobacteria, and of the utilization of the response to salt stress as a factor that can stabilize production in a cyanobacterial cell factory.</jats:p>

Heterologous production of glycine betaine using Synechocystis sp. PCC 6803-based chassis lacking native compatible solutes

“…As predicted by the metabolic flux model, the device may drain the cell's resources imposing a metabolic burden, causing growth impairment. This can be explained by the redirection of part of the photosynthetically fixed carbon to the synthesis of CS, which is no longer available for biomass formation, similarly to what was reported for the production of mannitol (55). In contrast to what was observed for the WT, the introduction of the GB device into the ΔggpS mutant resulted in an increased salt tolerance with the concomitant growth improvement, enabling its survival under 5% NaCl.…”

Integrative analysis of the salt stress response in cyanobacteria

“…Furthermore, salt acclimation is becoming more important for applied research with cyanobacteria regarding the direct use of compatible solutes as well as mass cultivation in sea water to make the process more sustainable (Pade and Hagemann, 2014; Cui et al, 2020). For example, the cyanobacterial production of mannitol (Wu et al, 2020) and trehalose (Qiao et al, 2020) has been promoted by cultivation at enhanced salinities. Moreover, a more salt-tolerant version of the fast-growing Synechococcus elongatus strain UTEX2973 has been engineered by the expression of GG synthesis genes, which can be used for biotechnological purposes in full marine waters (Cui et al, 2021) However, saline conditions might also negatively affect the production titer.…”