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

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.

Genomic Characterization of Non-Mucus-Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis

“…We selected random chemical mutagenesis with ethyl methanesulfonate (EMS), as it is an alkylating agent known to cause mainly transition mutations (27,28). EMS and other mutagens are used routinely in industrial settings to enhance the qualities of industrial microbial strains (28)(29)(30)). An enrichment method was established in which an excess of immobilized anti-SpaC IgG was used to trap piliated L. rhamnosus GG cells while nonpiliated mutants remain unbound.…”

Alcohol‐tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single‐cell mutant screening system

“…Tillich et al (2012) showed that the UV-C dosage giving a survival rate of ca. The survival rates of cells were 100.0 AE 5.6% (for no irradiation), 20.8 AE 4.7% (for 15 min irradiation), 5.7 AE 1.6% (30 min), 4.4 AE 0.3% (45 min), 0.7 AE 0.1% (60 min), and 0.0 AE 0.0% (90 min).…”

“…The exposure time for mutagenesis was adjusted so that the survival rate was about 10% (Tillich et al, 2012). The plate was irradiated with UV-C using a sterilization lamp (lmax ¼ 253.7 nm, GL-15, Hitachi Appliances, Inc.,) at a distance of 70 cm.…”

Engineering cyanobacteria for industrial products

“…Increases of stress tolerance against, for example, temperature (Tillich et al, 2012) or salinity (Bagchi et al, 2007;Ruffing, 2011) not only leads to enhanced longevity, but may also serve to protect cyanobacterial cultures against contamination with other (heterotrophic) microorganisms, as happens for, for example, Arthrospira mass cultivation.…”