Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides
© Koh et al.; licensee BioMed Central Ltd. 2014
Received: 29 November 2013
Accepted: 21 February 2014
Published: 27 February 2014
Rhodosporidium toruloides is a β-carotenoid accumulating, oleaginous yeast that has great biotechnological potential. The lack of reliable and efficient genetic manipulation tools have been a major hurdle blocking its adoption as a biotechnology platform.
We report for the first time the development of a highly efficient targeted gene deletion method in R. toruloides ATCC 10657 via Agrobacterium tumefaciens-mediated transformation. To further improve targeting frequency, the KU70 and KU80 homologs in R. toruloides were isolated and characterized in detail. A KU70-deficient mutant (∆ku70e) generated with the hygromycin selection cassette removed by the Cre-loxP recombination system showed a dramatically improved targeted gene deletion frequency, with over 90% of the transformants being true knockouts when homology sequence length of at least 1 kb was used. Successful gene targeting could be made with homologous flanking sequences as short as 100 bp in the ∆ku70e strain. KU70 deficiency did not perturb cell growth although an elevated sensitivity to DNA mutagenic agents was observed. Compared to the other well-known oleaginous yeast, Yarrowia lipolytica, R. toruloides KU70/KU80 genes contain much higher density of introns and are the most GC-rich KU70/KU80 genes reported.
The KU70-deficient mutant generated herein was effective in improving gene deletion frequency and allowed shorter homology sequences to be used for gene targeting. It retained the key oleaginous and fast growing features of R. toruloides. The strain should facilitate both fundamental and applied studies in this important yeast, with the approaches taken here likely to be applicable in other species in subphylum Pucciniomycotina.
Rhodosporidium toruloides is a β-carotenoid accumulating oleaginous yeast in subphylum Pucciniomycotina. Able to accumulate more than 70% of its dry cell mass as triacylgleride with similar chemical composition to those of plants from ultra-high density fermentation [2–4], R. toruloides is regarded as a great host with vast biotechnological potential to produce single cell oil, which may find wide spread applications in staple food, animal feed, biodiesel, surfactant and raw material for industrial polymers [3, 5]. Although studies have been done to optimize lipid yield through high-density fermentation , there are scarce reports on the rational genetic engineering to improve lipid accumulation or fatty acid profiles in R. toruloides. To date, there are no reverse genetic studies reported in R. toruloides. With the advent of efficient and stable transformation method established using Agrobacterium tumefaciens-mediated transformation (ATMT) in R. toruloides, reverse genetic studies should become a real possibility.
Targeted gene deletion, often referred as targeted gene knockout, is an essential tool for genetic engineering and reverse genetics. This is an important cornerstone to make any strains commercially competitive . While targeted gene integration in model microorganisms, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, can be done with ease and high efficiency [8, 9], it is a major obstacle in many industrially important species such as R. toruloides.
It has been proposed that DNA repair of double-stranded breaks by homologous recombination (HR) and non-homologous end-joining (NHEJ) operate competitively , and the predominance of NHEJ over HR has been regarded as the main cause of low gene targeting efficiency in fungi [11, 12]. Correspondingly, one strategy to deal with low gene targeting efficiency in fungi is to improve the HR pathway [11, 13]. The other strategy is to inhibit or eliminate the NHEJ pathway, thereby forcing the transformed DNA to be integrated via HR. With this approach, the frequency of HR has been found to be significantly improved with many reports of success in recent years through the disruption of NHEJ pathway by deleting one or more of its key components . In eukaryotes, the main component of the NHEJ system is the DNA-dependent protein kinase (DNA-PK), a three-protein complex consisting of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the regulatory DNA-binding subunits, the Ku70/80 heterodimer . The Ku heterodimer is an abundant nonspecific DNA-binding protein comprising of two tightly-associated subunits of about 70 and 83 kDa, named Ku70 and Ku80 respectively . Both proteins exist in organisms ranging from fungi to human, and are arguably the defining proteins of NHEJ because of their sequence conservation .
Here, we report the isolation and characterization of KU70 and KU80 homologs in R. toruloides and the evaluation of a KU70-deficient mutant strain generated for improving gene deletion efficiency in R. toruloides.
Isolation and characterization of Ku70 and Ku80 encoding genes in R. toruloides
Comparison of KU70 / 80 organization between fungal homologues
GenBank accession no.
CDS CG (%)
Intron CG (%)
Average intron length (nt)
Targeted gene deletion in wild type R. toruloides and generation of KU70 null mutants
Gene deletion frequency was improved in the ∆ku70 mutant
Gene deletion frequency in WT and ∆ku70e strains
Homolgy lengtha (bp)
Gene deletion frequencyb
Effect of homology sequence length on deletion frequency
Effects of homologous sequence length on CAR2 deletion frequency
Homology length (bp)
Gene deletion frequencya
Sensitivity of KU70 deficient mutant to DNA damaging agents
With more than 60% GC content, the KU70 and KU80 characterized here present the most GC-rich genes in the NHEJ-pathway reported so far. In terms of gene structure, both genes contain much higher density of introns than those of Y. lipolytica (Table 1), which is the best-studied oleaginous yeast to date. Not surprisingly, homologues of C. neoformans, which is under the same Basidiomycota phylum, also have high density of introns (Table 1).
DSB repair can differ in heterochromatic and euchromatic regions of the genome and histone modifying factors play an important role in this process [28, 29]. Recombination frequencies are known to vary in different genes even when assayed with the same technique and in the same genetic background . Impairment of the NHEJ-pathway has proved to be effective in improving homologous recombination frequency in many eukaryotic hosts. However, the magnitude of improvement appears to vary considerably in different reports. With a homology sequence of approximately 750 bp, the CAR2 deletion frequency was improved 7.2-fold, from 10.5%, in WT to 75.3% in the KU70-deficient mutant in R. toruloides. This is similar to the deletion of TRP1 in Y. lipolytica although substantially higher knockout frequencies have been reported for several genes in other fungi, for example, N. crassa, A. niger and C. neoformans (Additional file 4). Nevertheless, the R. toruloides STE20 gene remained very difficult to knockout even with the ∆ku70e mutant (Table 2). This demonstrates a positional effect and implies additional factors that regulate gene deletion in R. toruloides. As the STE20 gene is located between the mating type loci RHA2 and RHA3 in R. toruloides, it is possible that the gene is within a transcriptionally silenced chromatin as was reported for the mating type genes in a number of other fungi [31, 32]. The low deletion frequency of STE20 suggests a potential role of chromatin structure and/or gene expression level in regulating DNA recombination in R. toruloides.
One of the drawbacks of NHEJ-deficient strains is its elevated sensitivity to DNA damage and the possibility of generating unwanted mutations . Indeed, the KU70-deficient strain studied here showed increased sensitivity to MMS and UV radiation. However, the mutant did not show severe growth defects under normal growth conditions. With comparable sugar consumption rate and fatty acid profile to the WT, the ∆ku70 and ∆ku70e strains should maintain much of the appeal of R. toruloides in industrial applications.
The KU70-deficient mutant generated herein was found to be effective in improving gene deletion frequency and retained the key oleaginous and fast growing features of R. toruloides. The strain should facilitate both fundamental and applied studies in this important yeast, with the approaches taken here likely to be applicable in other species in subphylum Pucciniomycotina.
Strains, media, and culture conditions
R. toruloides strain ATCC 10657 and ATCC 204091 (previously named Rhodotorula glutinis) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured at 28°C in YPD broth (1% yeast extract, 2% peptone, 2% glucose, w/v) or on potato-dextrose agar (PDA). A. tumefaciens strain AGL1  was grown at 28°C in either liquid or solid 2YT medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.5). Escherichia coli XL1-Blue was cultured at 37°C in Luria-Bertani (LB) broth or on LB agar for routine recombinant DNA work.
Rapid amplification of cDNA ends (RACE)
Sequence (5′ to 3′)
Sense primer for KU70 3′ RACE Specific primer for ∆ku70 fungi colony PCR
Antisense primer 1 for KU70 5′ RACE
Antisense primer 2 for KU70 5′ RACE
Antisense primer 3 for KU70 5′ RACE
Sense primer for KU80 3′ RACE
Antisense primer 1 for KU80 5′ RACE
Antisense primer 2 for KU80 5′ RACE
Antisense primer 3 for KU80 5′ RACE
KU70 gene deletion region
CAR2 deletion region (50 bp homology length)
CAR2 deletion region (100 bp homology length)
CAR2 deletion region (250 bp homology length)
CAR2 deletion region (500 bp homology length)
CAR2 deletion region (750 bp homology length)
CAR2 deletion region (750 bp homology length) CAR2 complementation region
CAR2 deletion region (1000 bp homology length)
CAR2 deletion region (1500 bp homology length) CAR2 complementation region
CAR2 deletion region (1500 bp homology length)
STE20 deletion region
URA3 deletion region
Specific primer for ∆ku70 fungi colony PCR
Specific primer for ∆car2 fungi colony PCR
Specific primer for GPD1 (reference gene) in multiplex PCR
KU70 probe for Southern blot
CAR2 probe for Southern blot
All restriction and modification enzymes used were from New England Biolabs Inc. (NEB, Ipswich, MA, USA), unless otherwise stated. Plasmid pEX2 is a pPZP200 derivative routinely used as the binary T-DNA vector backbone .
pEX2 was digested with Sac I (blunt-ended) and Pme I to remove the hygromycin resistant gene cassette (Pgpd::hpt::T 35S ), and inserted with the 3,618 bp 5′-phosphorylated KU70 DNA fragment amplified from genomic DNA of R. toruloides ATCC 10657 using oligos Rg70Lf and Rg70Rr to create pEX2KU70. pDXP795hptR contained a hygromycin selection cassette composed of the endogenous GPD1 promoter from R. toruloides (795 bp version), codon-optimized hygromycin phosphotransferase gene (JQ806387, hpt-3) and the terminator of nopaline synthase gene of A. tumefaciens (P GPD1 ::hpt-3:: T nos , Additional file 5A), and the selection cassette is flanked at both ends by loxP sites, allowing its deletion by Cre recombinase that can be activated when required (Liu et al., unpublished data). To create KU70 deletion vector pKOKU70, hygromycin selection cassette was digested from pDXP795hptR by Bam HI-Hin dIII and the blunt-ended fragment was inserted into Sma I-Sac I (blunt-ended) sites of pEX2KU70. Similarly, pKOCAR2 was constructed first by cloning the 2,697 bp 5′-phosphorylated CAR2 fragment into pEX2, which was amplified using oligos Rt079 and Rt080. Subsequently, the P GPD1 ::hpt-3::T nos cassette was inserted between Sac II-Apa I (blunt-ended) sites, generating pKOCAR2 containing a homologous flanking sequence of approximately 750 bp at both ends. Gene deletion vectors for CAR2 carrying homology sequence of various lengths (50, 100, 250, 500, 750, 1000 and 1500 bp) were likewise constructed using oligonucleotides listed in Table 4 (Additional file 2). For CAR2 complementation, a 3,242 bp fragment amplified by oligos C1500f and Rt080 was 5′-phosphorylated and inserted to Hin dIII digested and blunt-ended pDXP795hptR to generate the complementation plasmid (Additional file 5B).
Using the same strategy for gene deletion vectors, the deletion region of STE20 and URA3 were amplified using oligos STE20Lf/STE20Rr (2,196 bp) and Rt33/Rt34 (2,784 bp), cloned into pEX2 and digested using Bsp HI/Nco I and Stu I/Mfe I (blunt-ended) to create pKOSTE20 and pKOURA3, respectively.
Transformation and identification of transformants
ATMT and fungal colony PCR were both performed as described previously . For further identification of gene deletion mutants, multiplex PCR  using genomic DNA as the template was performed to prevent false negative results. Two sets of primer pairs, one specific to the deletion target (Rg70f3/Rg70r2 and Rt096/Rt097 for KU70 and CAR2 gene, respectively) and the other to the reference gene GPD1 (Rt006 and Rt007) were added to the reactions.
Isolation of genomic DNA, RNA and Southern blot analysis
Cell cultures at exponential stage were collected and genomic DNA was extracted using MasterPure™ Yeast DNA purification kit (Epicentre, Madison, WI, USA), while RNA was extracted as described previously . The concentrations of extracted DNA or RNA samples were determined with NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and their integrity were checked by agarose gel electrophoresis.
For Southern blot analysis, 10 μg of genomic DNA was digested with Pvu I at 37°C for about 24 hrs and resolved by electrophoresis in a 0.8% agarose gel. Southern hybridization and detection procedures were performed using DIG (digoxigenin)-High Prime DNA Labeling and Detection kit in accordance with the manufacturer’s instructions (DIG Application Manual for Filter Hybridization, Roche Diagnostics, Indiana, IA, USA). The probes were amplified by PCR labeling using DIG DNA labeling mix, with primers Rt100 and Rt101 used to amplify a fragment targeting the 5′ flanking sequence of KU70, and Rt083 and Rt084 specific to the 5′ flanking sequence of CAR2.
Sensitivity to DNA-damaging agents
MMS and UV radiation were the DNA-damaging agents used to analyze strain sensitivity monitored by spot plate assay. Cell cultures in YPD broth were adjusted to one OD600 unit and 10-fold serial diluted, from which the diluted samples were spotted on YPD agar plates supplemented with MMS (Sigma, MO, USA) ranging from 0.001-0.1%. Exposure to UV radiation was done by placing the plates in a UV Crosslinker (Spectrolinker™ XL-1000, Spectronics Corporation, NY, USA) at a dose ranging from 100 to 600 J/m2 after the samples were spotted.
Freshly cultured cells were analyzed using a Nikon Eclipse 80i microscope equipped with CFI Plan Apochromat objectives (Nikon, Melville, NY, USA). Images were acquired with a Nikon DS camera interfaced with NIS-Element F 3.0 software.
GenBank accession numbers
The annotated KU70 and KU80 sequences from R. toruloides ATCC 204091 have been deposited in GenBank under the accession number of KF850470 and KF850471, respectively.
This material is based on research supported in part by the Singapore National Research Foundation under CRP Award No. NRF-CRP8-2011-02, the Singapore Economic Development Board and Temasek Trust. We thank Professor Mark Featherstone, Nanyang Technological University, Singapore, for the kind discussions of the work.
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