Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides

Abstract

The oleaginous yeast Rhodosporidium toruloides is an unconventional yeast species that can accumulate a high content of lipids. Because it belongs to the basidiomycetous group of fungus, limited tools and functional elements are available for genetic engineering of R. toruloides and related red yeasts. Here we report the functional evaluation of five constitutive promoters from this yeast. We assembled a reporter gene expression cassette, consisting of a promoter, the hygromycin gene (HYG) and the nos terminator, and inserted it into the binary vector pZPK. Hygromycin-resistant transformants were obtained when R. toruloides cells were co-cultured with Agrobacterium tumefaciens AGL1 cells harbouring the engineered vector. Genomic integration of the reporter cassette was verified by successful amplification of target DNA fragments. Quantitative PCR analysis suggested that the transformant had only one copy of the reporter cassette. The strength of these promoters was demonstrated at the phenotypic level on the hygromycin-gradient plate and at the transcriptional level by real-time quantitative PCR. It was found that the strengths of these promoters varied no more than five-fold and followed a decreasing sequence of PPGI, PPGK, PFBA, PTPI, and PGPD. This study established new genetic elements for the construction of superior R. toruloides strains to produce advanced biofuels and related chemicals.

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species

“…Therefore, promoters of genes involved in various steps of the pathway are particularly useful. Such examples include GPD1 , PGK1 and PGI1 promoters as reported previously [7, 11]. Here, we analyzed the promoters of 6 genes involved in either fatty acid biosynthesis, TAG biosynthesis or lipid body formation.…”

Improvement of lipid production in oleaginous yeast Rhodosporidium toruloides by ultraviolet mutagenesis

“…First-strand cDNA was synthesized using a PrimeScript TM II First-strand cDNA Synthesis Kit (TaKaRa) and adopted as a template for reverse transcription PCR amplification. The -actin gene was used as an internal reference gene for all experiments [25]. The primers for RT-qPCR were designed using Primer Premier 5.0 software and are listed in Supporting Information Table 1.…”

“…RT-qPCR analysis of genes was performed using a LightCycler® 480 Real-Time PCR System with SYBR Green Real-Time PCR Master Mix (Toyobo). The -actin gene was used as an internal reference gene for all experiments [25]. The expression levels of the genes were calculated using the 2 −ΔΔCt method.…”

A toolset of constitutive promoters for metabolic engineering ofRhodosporidium toruloides

“…A growing branch of synthetic biology involves the pursuit and improvement of standard parts that are reliable, orthogonal and robust for non-conventional hosts [8,9]. A small selection of promoters has already been characterized to modulate expression of heterologous genes in R. toruloides, the majority of them being metabolite-responsive promoters rather than constitutive [7,[10][11][12][13]. While promoters that are responsive to certain metabolites are valuable tools, a toolset of well characterized constitutive promoters remains necessary to explore the full potential of strain engineering.…”