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 is high has been widely used in bacteria yeast. In comparison with CRISPR–Cas9, CRISPR–Cas12a many outstanding advantages. Here, we report an Acidaminococcus sp. BV3L6 (As) Cas12a-based genome-editing for Starmerella bombicola. To demonstrate the CCMGE system, verified a counter-selectable S. bombicola, orotidine 5’-phosphate decarboxylase (100% URA3). We also tested common gene UDP-glucosyltransferase UGTA) using 300 bp donor containing hygromycin expression cassette. This toolkit was further extended to simultaneously edit two genes (18% UGTA leu) three (13.8% UGTA, leu system greatly reduces screening time multi-site editing. Based on PHA (polyhydroxyalkanoate)-producing strain constructed by increasing copy number synthase (PHAC). content DCW reached 11.8% 30.1 g/L, respectively. yield about times higher than that single-copy same fermentation method.