Minos And Restless Derived Transposon Insertion Mutagenesis Of Polar Psychrophilic Fungus And Screening Of Normal Temperature Adaptive Mutants

The polar regions have a wide variety of various microorganisms.They survive in extremely harsh polar regions with unique adaptation mechanisms and physicochemical properties.The extreme environment promotes them producing natural products with novel structures and good activity.However,the current understanding,development and utilization of polar microorganisms are still insufficient.Industrial application of these microorganisms and their products is hardly achieved because of their cold-adaptive production characteristics.Geomyces sp.WNF-15A,as an Antarctic psychrotrophic filamentous fungus,can secrete natural red pigments of high stability,good acid-base tolerance,safety and non-toxicity,and high color value.It holds good potential to be used as edible pigments and is being applied for new food additive in China.However,this fungus can only produce red pigments at low temperatures(13～15℃),limiting its large-scale industrial applications.In this study,positive mutants of Geomyces sp.WNF-15A were obtained by transposon insertion mutation method,allowing the red pigments produced at 25℃.Firstly,four transposable systems of Minos and Restless elements were established.Endogenous transposase of Minos and Restless elements were not identified in Geomyces sp.WNF-15A by sequence alignment and PCR cloning.Then two transposon systems of pFC000MH and pFC000-RH were successfully constructed.Also,two dual systems were also established with the donor plasmid pFC-MD and the helper plasmid pFC-MH of the Minos element and the donor plasmid pFC-MH and the helper plasmid pFC-RH of the Restless element.Secondly,the four constructed transposable systems were introduced into Geomyces sp.WNF-15A.Results indicated that the dual systems were more dominant.The conversion efficiency of the dual systems reached 25.9%for Minos and 12.4%for Restless,and the transposable efficiency of them was 11.9%and 13%,respectively.However,the positive mutation rate of the same transposon elements in the two different systems was almost the same,with the Minos element of～11%and the element of～10%.Finally,with four transposable systems 23 positive mutants with red pigments production and 6 positive mutants in growth were verified from solid plate culture.Production of red pigments from these mutants were further tested in liquid fermentation.At 14℃,the MPS1 achieved the highest level of red pigments(OD520=43.3),which was 78.4%higher than the wild-type strain.When cultured at 20℃,the production of MPS1 were still the highest(OD520=29.7),which was 128.7%higher than the wild-type strain.At 25℃,production of red pigments of reached the highest level in MPS3(OD520=5.3),followed by MPS1(OD520=5),MPD1(OD520=4.9)and MPS4(OD520=4.7).In contrast,the wild-type strain had no red pigments synthesis at this temperature.In conclusion,Minos and Restless transposon insertion mutation systems for polar psychrotrophic fungi were successfully developed in this study.Mutants that can produce highlevel red pigments at low temperature were obtained.Also,mutants that can produce red pigments at room temperature were successfully obtained,which broke through the restriction that the wild-type strains cannot synthesize red pigments at room temperature.Our study also provides new ideas and methods for mutation screening of other fungi from extreme environments such as polar and deep sea.