Novel centromeric plasmid for stable extrachromosomal gene expression in Aurantiochytrium limacinum

Abstract: Aurantiochytrium limacinum holds great promise for producing sustainable single-cell oil as an alternative to fish oil. However, research into its complex biological and biochemical characteristics and efforts toward strain improvement have been hampered by insufficient genetic tools. Until now, genetic transformations of A. limacinum have relied solely on chromosome integration, which is inefficient and prone to insertional mutagenesis and other issues related to genetically modified organisms (GMOs). This paper describes the first centromeric plasmid for A. limacinum. Amplification of this shuttle vector by E. coli enables direct delivery into A. limacinum via electroporation, where it undergoes stable replication and segregation into daughter cells. The key to the stable plasmid maintenance lies in a 500 bp segment derived from chromosome 24 of Phaeodactylum tricornutum. While this segment does not significantly enhance the efficiency of vector transformation, it enables the replication and maintenance of the shuttle vector in the host cell as closed circular DNA. The plasmid from three transformants demonstrates a high segregation efficiency of 96.8 ± 0.3% (n = 3), even in the absence of antibiotic selection. This novel centromeric plasmid considerably enhances the flexibility of genetic manipulations and gene expression in A. limacinum, opening new avenues for its study and industrial application. Key points: • First centromeric plasmid developed for genetic transformation in A. limacinum. • The novel plasmid enhances flexibility in genetic manipulation and gene expression. • The plasmid achieves 96.8 ± 0.3% (n = 3) segregation efficiency without antibiotic selection.