| Astaxanthin(AX),also known as astaxanthin,is a terpene compound.Because of its special structure,it has strong antioxidant activity,which is 500 times that of vitamin E,so it is named "Super Vitamin E".After a large number of experimental studies and clinical data,researchers found that astaxanthin has good biological functions in anti-tumor,protecting eyes and central nervous system,resisting radiation,enhancing immunity,etc.In addition,it also has good coloring ability,and has a good application prospect in food,medicine and aquaculture industries.There are three main industrial production methods of astaxanthin:artificial extraction;Chemical synthesis;Microbial fermentation method.Xanthophyllomyces dendrorhous can synthesize astaxanthin by using various low-cost carbon and nitrogen sources.Although the astaxanthin production of wild-type Xanthophyllomyces dendrorhous is relatively low,its industrial production process will be greatly increased through genetic breeding.1)based on the comparative analysis of the existing transcripts of wild-type and astaxanthin-producing Xanthophyllomyces dendrorhous,three genes,THI4,cys C and Xden1,were identified as target genes.The expression of THI4,cys C and Xden1 decreased with the increase of astaxanthin production by reverse transcription real-time fluorescence quantitative PCR,which indicated that these three genes were related to astaxanthin.2)using CRISPR/Cas9 gene editing technology,three mutant strains were obtained,which were labeled as Stran1(M△THI4),Stran2(M△cys C)and Strain 3(M△Xden1).The yield of astaxin detected by HPLC was 17.83 μg/m L,20.19 μg/m L and 18.76 μg/m L.The results showed that the astaxanthin production of Stran1(M△THI4),Stran2(M△cys C)and Strain 3(M△Xden1)increased by 45.79 %,65.09 % and 54.42 % respectively compared with the original strain Stran M(astaxanthin production was 12.23 μg/m L).3)Through the reverse transcription real-time fluorescence quantitative PCR experiment of mutant strain and starting strain,it can be seen that after cys C knockout,the expressionlevels of THI4 and Xden1 are obviously down-regulated compared with that of starting strain.When THI4 was knocked out,the expression of cys C and Xden1 was down-regulated to some extent.The expression of THI4 and cys C was down-regulated when Xden1 was knocked out.The results show that if any gene is knocked out,the other two genes will be down-regulated,thus inferring that there is a certain correlation among the three genes.To sum up,based on the comparative analysis of existing transcripts of wild-type and astaxanthin-producing Xanthophyllomyces dendrorhous,this study selected three genes that may affect astaxanthin synthesis.Three mutant Xanthophyllomyces dendrorhous strains were obtained by editing these three genes with CRISPR/Cas9 gene editing technology.The astaxanthin content of three mutant strains was detected by high performance liquid chromatography.Finally,reverse transcription real-time fluorescence quantitative PCR was carried out on the three genes of the mutant strain,and it was found that knocking out any one gene would lead to down-regulation of the two genes,thus inferring that there was a certain correlation among the three genes. |
