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The Optimal Mutagen Dosage to Induce Point-Mutations in <em>Synechocystis</em> sp. PCC6803 and Its Application to Promote Temperature Tolerance

<div><p>Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria <em>Synechocystis</em> sp. PCC6803. Based on these results an optimal dosage of 10–50 J/m² for UV and either 0.1 or 1 v% for MMS was determined. A <em>Synechocystis</em> wildtype culture was then mutagenized and selected for increased temperature tolerance <em>in vivo</em>. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate <em>in vivo</em> screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for <em>in vivo</em>, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods.</p> </div

Summary of the conditions and results of each round of mutagenesis.

*<p>Note that in the 3rd round of mutagenesis there was a technical problem with the temperation, and cultures were also temperated during the night cycle, leading to many more cells dying than usual. A recovery at lower temperature was used to rescue surviving cells.</p

Characterization of UV.

<p>Survival rate (green; triangle) and point mutation rate (blue; square) plotted over the administered dosage of UV irradiation. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p

Characterization of MMS.

<p>Survival rate (green; upward triangle: 5*10∧4 cells plated | downward triangle 5*10∧5 cells plated, counted colonies divided by 10) and point mutation rate (red; square) plotted over the administered dosage of MMS exposure. The counted colonies for the survival rate and the point mutation rate were normalized by setting the control to 100%.</p