Established An Efficient CRISPR-iCas9 Gene Editing System And A Zeaxanthin Synthetic Strain In Yarrowia Lipolytica

In recent years,the use of microbial metabolism to produce target compounds has attracted widespread attention.Microorganisms can be transformed through gene editing technology to transform cheap carbon and nitrogen sources into a variety of high value-added products.Zeaxanthin is a fat-soluble terpene compound that has many biological effects,such as inhibiting tumor cells and preventing cataracts.Since the body cannot synthesize it,it must be obtained through dietary supplementation.Therefore,zeaxanthin has been widely concerned in the field of food and health products.Yarrowia lipolytica isgenerally recognized as a safe yeast strain.After genetic modification,it has been widely used in the production of fatty acid-derived biofuels and chemicals.However,the application of CRISPR-Cas9 technology in the genetic modification of Y.lipolytica is faced with the problems of low efficiency and low throughput.It is necessary to optimize the system to improve the efficiency of gene knockout and integration.Thus,this study established a highly efficient gene editing techniques CRISPR-iCas9,which can quickly achieve meaningless gene knockout and purpose of the integration.The CRISPR-iCas9achieves high efficiency and high flux gene editing effect.Its use in solving the exogenous synthetic zeaxanthin with access to build in Y.lipolytica,integrate zeaxanthin synthetic key genes to improve the target product yield.The research results are as follows:(1)Site-directed mutagenation of two amino acids in the REC1 recognition region,D147Y and P411T,was conducted to optimize the Cas9 domain and established the CRISPR-iCas9 system.The knockout efficiency of the CRISPR-iCas9 system was 100%,and the knockout efficiency of mfe1?gut2?pox3 and pex10 could reach 80%,indicating that the CRISPR-iCas9 system was widely applicable.Because the non-homologous end repair led to huge difference in the genetic sequence of the mutant strain,so a two-plasmid directed knockout system was designed to knock outgut2 and pox3.The sequencing results showed that the knockout was repaired according to Donor DNA sequence,including 23 bp deletes of sgRNA and PAM,and the result of the directed knockout was achieved with an efficiency of 100%.(2)One step integrate ?-carotene synthetic key genes carRA/carB/GGS1/tHMG usingCRISPR-iCas9,successfully constructed the?-carotene accumulated strain YL-ABTGto shake flask fermentation.The results showed that the strain growth in good condition,strain stability is strong,and the final?-carotene yield can reach 3.12 mg/g DCW.The CRISPR-iCas9 can be integrated with large fragment genes in chromosomes.(3)The zeaxanthin biosynthesis pathway was successfully built using CRISPR-iCas9,converting ?-carotene into higher-value products.The expression of?-carotene-3-hydroxy gene(CrtZ)can promote the zeaxanthin biosynthesis,the yield reach up to 2.33 mg/g DCW.In conclusion,the CRISPR-iCas9 system established in this study can shorten the time of genetic modification,and quickly realize the knockout of meaningless genes.Moreover,this technology can integrate large fragments of target genes into chromosomes and realize rapid one-step multi-gene integration.Therefore,CRISPR-iCas9 technology provides a feasible scheme for efficient genetic modification of Y.lipolytica strains,and provides effective support for increasing the yield of biological compounds and obtaining higher commercial value.