Abstract
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 Synechocystis 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 Synechocystis wildtype culture was then mutagenized and selected for increased temperature tolerance in vivo. 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 in vivo 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 in vivo, 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.
Highlights
There has been a revamped interest in algae and cyanobacteria biotechnology in recent years, mostly due to the possible applications for biofuel production [1,2,3].As experience has shown, it is very unlikely that any form of large scale cultivation will be performed using wildtype strains
5*107 cells/ml was placed on a petri dish, homogeneously spread, and irradiated with up to 300 J/m2 Ultraviolet light (UV) light
Irradiation was performed in a UVC 500 crosslinker (Hoefer, San Fransisco) with only one lamp (Sankyo Denki G8T5; 8W) for a more precise dosing at a wavelength of,254 nm
Summary
There has been a revamped interest in algae and cyanobacteria biotechnology in recent years, mostly due to the possible applications for biofuel production [1,2,3].As experience has shown, it is very unlikely that any form of large scale cultivation will be performed using wildtype strains. There has been a revamped interest in algae and cyanobacteria biotechnology in recent years, mostly due to the possible applications for biofuel production [1,2,3]. Most currently cultivated plants or animals have been extensively modified to be more suitable to application either through breeding, random mutagenesis (green revolution), and/or targeted approaches (modern GM crops). Random mutagenesis is a useful tool to adapt stains to cultivation conditions such as the induction of tolerances. It is possible to achieve effects which require simultaneous modifications of many seemingly unrelated genes, such as those required for increased tolerance to ethanol [4]. When choosing the proper agent a broad spectrum of mutations can be achieved
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