| The heterotrophic marine microalga Crypthecodinium cohnii can accumulate DHA that makes up nearly 30-50% of its constituent fatty acids,and meanwhile accumulates less than 1% of the other type of PUFAs.Due to these advantages,it has been widely used in industrial fermentation for algal DHA production in recent years.However,so far the whole genome sequence of C.cohnii has not been completed,which has redistricted the application of more radical genetic engineering approach to obtain C.cohnii mutants with better biological properties.Alternatively,high-yield mutants of C.cohnii can be obtained using suitable mutagenesis and high-efficiency screening.In the study,a high-concentration of sethoxydim was found to inhibit the C.cohnii growth significantly.We thus proposed to apply sethoxydim as a selective agent to screen mutants with increased ACCase activity,and possibly increased growth and lipid-content as well.Towards this goal,an integrated ARTP mutagenesis and sethoxydim-based screening protocol was applied to C.cohnii for two rounds,leading to the identification of several mutants with high growth and lipid-content.The efforts led to identification of a mutant M-1-2 with 24.32% higher growth and 7.05% higher lipid content than those of the wild type,respectively,demonstrating the effectiveness of the sethoxydim-based screening.Consistently,comparative ACCase enzymatic activity and transcriptomic analysis showed that the ACCase activity was increased by 16.15%,and the expression of ACCase encoding genes were up-regulated by 1.53 to 3.12 folds in the M-1-2 mutant,respectively.In addition,a transcriptomic analysis showed that transcripts involved in fatty acid biosynthesis,energy,central carbohydrate,and amino acid metabolism were also up-regulated in the mutant when compared with the wild type.The new information obtained could be useful for the better understanding of C.cohnii metabolism and for the engineering of the high-yield C.cohnii strains in the future. |
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