Developing a targeted gene deletion system in Rhodosporidium toruloides.

• Main Author: Koh, John Chong Mei.
• Other Authors: Mark Stephen Featherstone
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Microbiology::Microorganisms; DRNTU::Science::Biological sciences::Molecular biology
• Online Access: http://hdl.handle.net/10356/55394
• Institution: Nanyang Technological University

Rhodosporidium toruloides is a red, oleaginous yeast, belonging to the subphylem Pucciniomycotina and phylem Basidiomycetes. This microorganism has generated strong interest in the biofuel community due to its ability to accumulate as much as 70% of its dry weight as lipids under ultra-high density fermentation (>200g/L dry cell mass). However, the lack of reliable and efficient gene manipulation tools has been a major roadblock for its progress as a biotechnology platform. A gene transformation method using Agrobacterium tumefaciens-mediated transformation (ATMT) was established by our laboratory recently, but the targeted gene deletion system remained elusive. Here, we report the development of a targeted gene deletion method in R. toruloides ATCC 10657 by ATMT. CAR2, a gene encoding a bifunctional protein involved in carotenoid pigment biosynthesis was selected as the gene target. Successful deletion of CAR2 led to albino instead of red transformants, thus providing an excellent platform for the study of gene deletion efficiency in Rhodosporidium species. We characterized homologues of recombination related genes KU70/80 and RAD51 and generated a KU70 mutant by targeted gene deletion. The frequency of homologous recombination was found to increase markedly from around 5% in the wild type stain to over 90% in the KU70-deficient strain when homologous flanking sequences of about 750 bp were used. Other strategies explored include over-expression RAD51 and use of a DNA-dependent protein kinase chemical inhibitor, NU7026. The gene deletion frequency was enhanced from around 5% in wild type strain to just over 7% in the RAD51 over-expressing strain. However, NU7026 was found to have no effect on gene deletion frequency in R. toruloides. The studies in this work should become a major boost to the biotechnological development as well as gene functional studies in R. toruloides and many other red oleaginous yeasts.