Abstract
BackgroundRhodotorula 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.ResultsWe 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.ConclusionsT-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.
Highlights
Rhodotorula toruloides is an outstanding producer of lipids and carotenoids
Application of Agrobacterium tumefaciens-mediated transformation (ATMT) in Puicciniomycotina subphylum We have reported a reliable transformation protocol for R. toruloides American Type Culture Collection (ATCC) 10657 (Rt1) using the dominant selection conferred by the codon-optimized hygromycin resistance gene [16]
While the method was generally applicable in several species or strains in Pucciniomycotina, e.g. R. toruloides ATCC 10788 (Rt2), R. glutinis ATCC 90781 (Rg1), R. glutinis ATCC 204091 (Rg2), R. graminis WP1 (Rg3), and Sporobolomyces roseus FGSC 10293 (IAM13481, Sr) (Fig. 1a), large variations in the transformation efficiency (TFE, or Colony Forming Unit per 106 fungal cells), were observed (Fig. 1b)
Summary
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. A large number of oleaginous microorganisms capable of producing more than 20% of their dry biomass as lipids have been reported to date [1,2,3] They are potential alternative hosts to plants for the production of lipid and fatty acid derivatives, such as biodiesel, alkane, fatty alcohol and wax [1, 4,5,6,7]. Information regarding the molecular control of metabolism and catabolism remains rare in this host, severely limiting the development of R. toruloides as an industrial workhorse. Chemical mutagens and ultraviolet radiation are often used to improve strains or populations of interests under a specific selection pressure Such techniques usually produce mutants with point mutations. Agrobacterium tumefaciens-mediated transformation (ATMT) delivers the T-DNA into the host’s nuclear genome and has been widely used as an IM tool, in fungi and plants [42,43,44]
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