| Starmerella bombicola is a kind of unconventional yeast.It has the ability to utilize oil,resist sugar and acid,and efficiently produce sophorolipids.It has a great potential for industrial application.The lack of efficient genetic transformation technology hinders the transformation of systemic metabolic engineering.The development of efficient gene editing technology can provide a solid technical support for the metabolic transformation of S.bombicola.In this study,the CRISPR-Cas12a gene editing system was constructed and the gene editing efficiency was evaluated for the first time in S.bombicola,and an engineering strain for the synthesis of PHA was constructed using this technology.The main results are as follows:（1）The construction of CRISPR-Cas12a gene editing system.Cas12a gene from Acidaminococcus sp.BV3L6 was optimized according to codon preference of S.bombicola.By homologous recombination,Cas12a expression cassette and hygromycin pop-up system expression cassette was expressed in S.bombicola,and the strain integrating Cas12a gene at sble site was obtained.Comparing the growth of this strain with that of wild type strain,the results showed that Cas12a gene had no toxic effect on the growth of the strain.By designing the guide RNA（cr RNA）targeting the coding region of leu,the plasmid containing the expression frame of cr RNA was constructed,and the linearized plasmid was transformed into S.bombicola.We found that Cas12a nuclease could cleave the target gene and perform NHEJ repair in the absence of repair template,proving that Cas12a nuclease had the cleavage function.When the length of the used repair template homologous arm is short,the editing efficiency is low,and when the repair template with the 300-bp homologous arm is used,the editing efficiency greatly improved.（2）The CRISPR-Cas12a gene editing system is based on the evaluation of editing efficiency of single gene and multiple genes.By using the CRISPR-Cas12a gene editing system,the editing efficiency of orotate nucleoside 5’-phosphate decarboxylase gene URA3 was 100%.At the same time,it is verified that the editing efficiency of non-marker gene UGTA is 100%in S.bombicola.Based on the ability of Cas12a nuclease to simultaneously target multiple genes with cr RNA array,the kit was further extended to simultaneously edit two genes（18%for UGTA and leu）and three genes（13.8%for UGTA,leu and URA3）.The system greatly reduces the screening time of the multi-site editing.（3）Construction of high-yield PHA engineering strain based on CRISPR-Cas12a.The peroxisome localization signal peptide of MFE2 from S.bombicola was fused with GFP,and GFP without localization signal peptide was used as control group.By comparing the distribution of GFP in cells,it is proved that the localization signal peptide has the function of localization in peroxisome The PHA-producing strain was constructed by increasing the copy number of the PHA synthase gene（PHAC）.The PHA content and dry cell weight（DCW）of the three-copy strain were 11.8%and 30.1 g·L-1,respectively,and the yield of PHA was about three times higher than that of the single-copy strain.Besides,the addition amount of rapeseed oil in the original fermentation medium was optimized.With PHA03 strain as the starting strain,when the addition amount of rapeseed oil was 6 m L（500 m L for fermentation flask and 100 m L for fermentation broth）,the PHA content and DCW reached 15.2%and 28.2 g·L-1,respectively,and the PHA yield was increased by about 28.8%. |
