Establishing An Adaptive Laboratory Evolution Method And Genetic Transformation System For Crypthecodinium Cohnii

The heterotrophic marine microalgae Crypthecodinium cohnii can accumulate 30%-50%DHA in its total lipid,while other type of PUFAs is less than 1%.Due to thses remarkable advantages,C.cohnii has been widely usded in the industry.Improvement of the productivity of the total lipids and DHA in C.cohnii will play significant roles in enhancing the supply of DHA.Due to the large genome and complexity,the C.cohnii genome sequence is not available yet,which restricts exploration of the mechanisms of lipid accumulation in C.cohnii.Meanwhile,it is impossibile to conduct genetic manipulations in C.cohnii to enhance its charactristics,because no valid transformation system is available.In this study,we developed and used a novel method which was termed“chemical modulators based adaptive laboratory evolution,to enchance the productivity of total lipids in C.cohnii.In addition,a transformation protocol was also estabolished which could be applied to systemically genetic engineer C.cohnii in the near further.We applied sethoxydim that targets acetyl-CoA carboxylase as a selective pressure for the first-round CM-ALE.In the E-S strain,50%carbon equivalents from starch were redirected to lipid.Sesamol,a kind of antioxidants,has been reported to enhance the biomass productivity in C.cohnii.In order to dress the dilemma of different effects of sesamol on lipid content and cell growth,sesamol based CM-ALE was conducted to release the sensitivity of sesamol to C.cohnii.With the addition of 1 mM sesamol in E-D,the productivity of lipid and DHA were increased by 100%and 130%,respectively.In this study,FITC-Dextran was used as detecting marker to evaluate the electroporation parameters for C.cohnii,and the results indicated that under the optimized conditions 2.0 kV,200?,50 ?F,the macromolecules could be successfully delivered into the C.cohnii cell.With using this system,we constructed a RuBisCO-deleted strain.Our results show that deletion of the RuBisCO gene promoted cell growth and increased lipid content in C.cohnii under heterotrophic growth conditions.