The traditional homologous recombination(HR)gene-editing method faces problems such as low editing efficiency and absence of marker genes.CRISPR-Cas9-editing efficiency is high and has been widely used in bacteria and...The traditional homologous recombination(HR)gene-editing method faces problems such as low editing efficiency and absence of marker genes.CRISPR-Cas9-editing efficiency is high and has been widely used in bacteria and yeast.In com-parison with CRISPR-Cas9,CRISPR-Cas12a has many outstanding advantages.Here,we report an Acidaminococcus sp.BV3L6(As)Cas12a-based genome-editing method used for Starmerella bombicola.To demonstrate the high efficiency of the CCMGE system,we verified a counter-selectable marker in S.bombicola,orotidine 5’-phosphate decarboxylase(100%for URA3).We also tested the common gene UDP-glucosyltransferase(100%for UGTA)using a 300 bp donor containing hygromycin expression cassette.This toolkit 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 for such multi-site editing.Based on the CCMGE system,the PHA(polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase(PHAC).The PHA content and DCW reached 11.8%and 30.1 g/L,respectively.The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.展开更多
基金The work was supported by the 111 Project(No.111-2-06)National Natural Science Foundation of China(32001064)+2 种基金Key Research and Development Program of China(2021YFC2100102-03)China Postdoctoral Science Foundation(2020M671331)Postgraduate Research and Practice Innovation Program of Jiangsu Province(No.KYCX20-1807).
文摘The traditional homologous recombination(HR)gene-editing method faces problems such as low editing efficiency and absence of marker genes.CRISPR-Cas9-editing efficiency is high and has been widely used in bacteria and yeast.In com-parison with CRISPR-Cas9,CRISPR-Cas12a has many outstanding advantages.Here,we report an Acidaminococcus sp.BV3L6(As)Cas12a-based genome-editing method used for Starmerella bombicola.To demonstrate the high efficiency of the CCMGE system,we verified a counter-selectable marker in S.bombicola,orotidine 5’-phosphate decarboxylase(100%for URA3).We also tested the common gene UDP-glucosyltransferase(100%for UGTA)using a 300 bp donor containing hygromycin expression cassette.This toolkit 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 for such multi-site editing.Based on the CCMGE system,the PHA(polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase(PHAC).The PHA content and DCW reached 11.8%and 30.1 g/L,respectively.The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.
