Deletion of the KU70 homologue facilitates gene targeting in Lipomyces starkeyi strain NRRL Y-11558

• Published in: Current genetics Vol. 65; no. 1; pp. 269 - 282
• Main Authors: Dai, Ziyu, Pomraning, Kyle R., Deng, Shuang, Hofstad, Beth A., Panisko, Ellen A., Rodriguez, Diana, Butcher, Mark G., Culley, David E., Magnuson, Jon K.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.02.2019; Springer Nature B.V
• Subjects: beta-glucuronidase; Biochemistry; Biodegradation; Biomedical and Life Sciences; Cell Biology; DNA Breaks, Double-Stranded - radiation effects; DNA End-Joining Repair - genetics; Efficiency; Fungal Proteins - genetics; Fungal Proteins - metabolism; Gamma Rays; Gene Deletion; Gene expression; Gene Expression Regulation, Fungal; gene overexpression; Gene sequencing; Gene targeting; Genes; Glycogen; Homologous recombination; Homology; Insertion; Ku Autoantigen - genetics; Ku Autoantigen - metabolism; Life Sciences; Lipids; Lipids - biosynthesis; Lipomyces - classification; Lipomyces - genetics; Lipomyces - metabolism; Lipomyces starkeyi; Loci; Microbial Genetics and Genomics; Microbiology; Mutagenesis, Site-Directed; Non-homologous end joining; Original Article; Overexpression; Plant Sciences; Proteomics; Reporter gene; reporter genes; Synthesis; Ultraviolet Rays; Yeast; yeasts; Non-homologous end-joining (NHEJ); Oleaginous yeast; Homologous recombination; Ls; Lipid production and degradation; LsKU70; Lipomyces starkeyi
• DOI: 10.1007/s00294-018-0875-z
• ISSN: 0172-8083; 1432-0983

The objective of this study was to disrupt the non-homologous end-joining (NHEJ) pathway gene (Ls ku70 Δ) and evaluate the effects of selected gene deletions related to glycogen synthesis (Ls GSY1 ) and lipid degradation (Ls MFE1 , Ls PEX10 , and Ls TGL4 ) on lipid production in the oleaginous yeast Lipomyces starkeyi . Disruption of the NHEJ pathway to reduce the rate of non-homologous recombination is a common approach used to overcome low-efficiency targeted deletion or insertion in various organisms. Here, the homologue of the Ls KU70 gene was identified and disrupted in L. starkeyi NRRL Y-11558. The Ls GSY1 , Ls MFE1 , Ls PEX10 , Ls TGL4 , and Ls URA3 genes were then replaced with a resistance marker in the Ls ku70 Δ strain and several site-specific insertions were assessed for targeted over-expression of selected genes. The targeted disruption efficiency of five selected genes (Ls GSY1 , Ls MFE1 , Ls PEX10 , Ls TGL4 , and Ls URA3 ) was increased from 0 to 10% in the parent to 50–100% of transformants screened in the Ls ku70 Δ strain with 0.8–1.4 kb homologous flanking sequences, while the efficiency of site-specific gene insertion with the β-glucuronidase reporter gene was 100% in the locus near the 3′-end coding (Ls KU70 ) and non-coding (Ls GSY1 , Ls MFE1 , and Ls PEX10 ) regions. Disruption of Ls KU70 in isolation and in conjunction with Ls GSY1 , Ls MFE1 , Ls PEX10 , or Ls TGL4 did not affect lipid production in L. starkeyi . Furthermore, β-glucuronidase reporter gene activity was similar in strains containing site-specific targeted insertions. Therefore, over-expression of genes related to lipid synthesis at targeted loci can be further examined for improvement of total lipid production in L. starkeyi .