Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

Abstract

Abstract:The Cre/loxP site-specific recombination system was applied to Aurantiochytrium limacinum to obtain a transformant without the antibiotic resistance marker gene. First, the enhanced green fluorescent protein gene (egfp) and chloramphenicol resistance gene (Cm r ), along with the two loxP loci, were integrated into the genome of A. limacinum OUC88 using 18S rDNA sequences as the homologous recombination sites. Then plasmid pSH65, containing a zeocin resistance gene (Ble r ) was transferred into A. limacinum OUC_CG. After induction with galactose, repeated passage in culture and PCR-based assessment, the pSH65 plasmid was lost and A. limacinum OUC_EG host was shown to no longer have resistance to 100 mg chloramphenicol/L or 5 mg zeocin/L. Through southern blotting and fluorescence detection, egfp was found to be integrated into the genome of A. limacinum OUC_EG, and EGFP was successfully expressed in the cells. The successful application of the Cre/loxP system demonstrates an experimental basis for genetic modification of A. limacinum so as to obtain transformed strains with no antibiotic resistance marker genes.

Cloning of the pks3 gene of Aurantiochytrium limacinum and functional study of the 3-ketoacyl-ACP reductase and dehydratase enzyme domains

“…The Cre/loxP site-specific recombination system has been applied to A . limacinum to obtain a transformant without the antibiotic resistance marker gene using 18Sr DNA sequences as the homologous recombination sites [17]. These studies have provided us with numerous genetic transformation methods.…”

“…Liquid medium [7% (w/v) glucose, 2% (w/v) yeast extract and 2% (w/v) sodium glutamate] with a salinity equivalent to 50% that of seawater was used for the propagation of A . limacinum [17].…”

“…Southern blotting was used to detect whether the gene was knocked-in following the procedures described by Sun et al[17]. The Yeast DNAiso Kit (Takara, Japan) was used to extract the genome DNA from A .…”

“…18S rDNA sequence as the homologous recombination site and PGK promoter and CYC1 terminator from Saccharomyces cerevisia as promoter and terminator were successfully applied to A . limacinum to express EGFP [17]. In this research, the kr and dh genes were knocked in A .…”

Alleviation of reactive oxygen species enhances PUFA accumulation in Schizochytrium sp. through regulating genes involved in lipid metabolism

“…Although metabolic engineering seemed an effective and promising way to alleviate ROS, no attempts have yet been made, to our knowledge, which demonstrates the effect of oxidative stress in thraustochytrids at the molecular level. Despite the knowledge of a versatile transformation system for thraustochytrids ( Hong et al, 2013 , Sakaguchi et al, 2012 , Sun et al, 2015 ), only a few studies were focused on the improvement of FA productivity through gene manipulation ( Table S1 ). Up to now, there are no comprehensive studies focused on ROS quenching to enhance PUFA productivity in thraustochytrids.…”