Screening And Mutation Breeding Of Oleaginous Filamentous Fungi

Screening oleaginous microorganisms with capability of accumulating considerable amounts of lipids is essential for industrial lipid production.Filamentous fungi have attracted more attention as it can produce various long-chain PUFAs.This study carried out a new screening strategy by using triphenyltetrazolium chloride(TTC,a redox indicator used for detecting oil)and cerulenin(an inhibitor of fatty acid synthase(FAS)).And potential strains were quickly isolated from microbial community by using this method.In order to further improve oil production of wild type strains,the potential strain was mutated with atmospheric and room temperature plasma(ARTP),and a high yield mutant strain with stable genetic stability was obtained combining with effective screening strategy.The main contents and results of this paper are as follows.1.Screening and identifying of oleaginous filamentous fungi.A new screening strategy was carried out by using TTC and cerulenin added in screening medium.According to the colony color(red colonies were selected)and diameter,we obtained 19 filamentous fungi isolates with high lipid productivity(>20% dry cell weight)from soil samples.The isolates of TSM-3 and SL-4 showed the better growth performance and lipid productity.Through the morphology identification and phylogenetic analysis based on ITS(Internal Transcribed Spacer)gene sequences,all 19 isolates were affiliated with the genus Mortierella.This study provides a new strategy to screen oleaginous filamentous fungi with more fast and efficient than traditional methods.2.The influence of different fermentation conditions on strain growth and lipid synthesis of candidate strains.Firstly,8 potential isolates were selected to study the effect of temperature for strains growth and lipid synthesis.Strains exhibited a broad growth temperature ranges from 4 to 28°C.The optimum growth temperature ranges distributed between 23? and 28?.Experiments confirmed that these isolates are also psychrotolerant,as they could still present slow-growing at 4°C.However,none of these isolates could grow at 37°C.Secondly,this study found that low temperature fermentation may promote PUFAs accumulation,and increased unsaturation of fatty acids.Furthermore,2 isolates with better growth and lipid-production characteristics were selected to study the effect of culture medium compositions on growth and lipid synthesis of the strains.The results showed that 2 isolates showed a wide range of carbon source utilization ability.Finally,it was found that using corn flour as carbon source and soybean meal as nitrogen source were beneficial for cell growth,and taking glucose as carbon source and yeast extract as nitrogen source were beneficial for lipid accumulation through studying the effects of different carbon and nitrogen sources on cell growth and lipid accumulation.3.The wild type strain TSM-3 was mutated by ARTP.The isolate of TSM-3 with better growth and lipid-production characteristics was selected as original strain for ARTP mutation.Combining with the effective screening strategy established in this study,a high yield mutant strain TSM-3-1 with genetic stability was obtained.Compared with the original strain TSM-3,the lipid and AA production of mutant TSM-3-1 increased 47.8% and 84.5%,respectively.