A CRISPR–Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola
Min Zhang, Yibo Shi, Lihua Zhang, Shiying Zhu, Haiquan Yang, Wei Shen, Yuanyuan Xia, Xianzhong Chen
Systems Microbiology and Biomanufacturing ›› 2022, Vol. 2 ›› Issue (4) : 665-675.
A CRISPR–Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola
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 comparison 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.
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