Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis

BMC Microbiology
Yanbin LiuLianghui Ji

Abstract

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. 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 an...Continue Reading

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Citations

Jul 3, 2020·FEMS Yeast Research·Zhiqiang WenZongbao K Zhao
Aug 25, 2018·Applied Microbiology and Biotechnology·Sujit Sadashiv Jagtap, Christopher V Rao
Apr 16, 2019·FEMS Yeast Research·Xiangjian LiuZongbao Kent Zhao
Mar 16, 2019·Current Opinion in Biotechnology·Michael SpagnuoloMark Blenner
Mar 21, 2021·Biotechnology for Biofuels·Yanbin LiuLianghui Ji
Dec 8, 2021·World Journal of Microbiology & Biotechnology·Yu ZhaoJianfa Zhang

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Methods Mentioned

BETA
PCR
electrophoresis
protein folding
reverse transcription PCR

Software Mentioned

hiTAIL
RQ Manager
BLASTn

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