Please use this identifier to cite or link to this item: https://doi.org/10.1186/s12866-018-1151-6
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dc.titleIdentification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis
dc.contributor.authorLiu, Y.
dc.contributor.authorKoh, C.M.J.
dc.contributor.authorYap, S.A.
dc.contributor.authorDu, M.
dc.contributor.authorHlaing, M.M.
dc.contributor.authorJi, L.
dc.date.accessioned2021-12-29T04:42:31Z
dc.date.available2021-12-29T04:42:31Z
dc.date.issued2018
dc.identifier.citationLiu, 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 (1) : 14. ScholarBank@NUS Repository. https://doi.org/10.1186/s12866-018-1151-6
dc.identifier.issn14712180
dc.identifier.urihttps://scholarbank.nus.edu.sg/handle/10635/212402
dc.description.abstractBackground: 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 single-copy 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. © 2018 The Author(s).
dc.publisherBioMed Central Ltd.
dc.rightsAttribution 4.0 International
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.sourceScopus OA2018
dc.subjectAgrobacterium tumefaciens-mediated transformation
dc.subjectCarotenoid and lipid biosynthesis
dc.subjectInsertional mutagenesis
dc.subjectMetabolic engineering
dc.subjectPucciniomycotina
dc.typeArticle
dc.contributor.departmentDEAN'S OFFICE (MEDICINE)
dc.contributor.departmentPHYSIOLOGY
dc.contributor.departmentBIOLOGY (NU)
dc.contributor.departmentINST OF MOLECULAR AGROBIOLOGY
dc.description.doi10.1186/s12866-018-1151-6
dc.description.sourcetitleBMC Microbiology
dc.description.volume18
dc.description.issue1
dc.description.page14
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