Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/89214
Title: Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis
Authors: Liu, Yanbin
Koh, Chong Mei John
Yap, Sihui Amy
Du, Minge
Hlaing, Mya Myintzu
Ji, Lianghui
Keywords: Agrobacterium Tumefaciens-mediated Transformation
Pucciniomycotina
Issue Date: 2018
Source: Liu, Y., Koh, C. M. J., Yap, S. A., Du, M., Hlaing, M. M., & Ji, L. (2018). Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis. BMC Microbiology, 18, 14-.
Series/Report no.: BMC Microbiology
Abstract: Background: Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. Results: We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly singlecopy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. Conclusions: T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.
URI: https://hdl.handle.net/10356/89214
http://hdl.handle.net/10220/44822
DOI: 10.1186/s12866-018-1151-6
Rights: © 2018 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Journal Articles

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