Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803

Abstract

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. PCC7002 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. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC6803 as a model cyanobacterium. Results: Here, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC6803, 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 utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

Comparison of alternative integration sites in the chromosome and the native plasmids of the cyanobacterium Synechocystis sp. PCC 6803 in respect to expression efficiency and copy number

“…While well-suited for comparing expression efficiencies in vivo, fluorescent markers offer very limited information on expression-related adverse effects, and the resulting host response towards negative selection. More severe metabolic stress is induced by expression systems that deplete the cell from essential metabolic precursors or enzyme cofactors [ 60 , 62 ], accumulate reactive pathway intermediates [ 63 ] or produce toxic end-products [ 64 ]. Such effects may be lethal or critically compromise the housekeeping functions of the cell, depending on the expression efficiency and the nature of the biochemical interactions, and must be assessed case-by-case when developing production strains for biotechnological use.…”

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 [ 9 , 68 ]. For example, the cyanobacterial production of mannitol [ 106 ] and trehalose [ 78 ] 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 [ 10 ].…”

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 (Wu et al, 2020). 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.…”