Polynucleotide sequences from rhodosporidium and rhodotorula and use thereof

The present invention relates to the application of isolated promoters and synthetic constructs for efficient production of genetically modified cells in a species selected from the Pucciniomycotina and Ustilaginomycotina subphyla, in particular, species selected from the Rhodosporidium, Rhodotourla, Sporobolomyces or Pseudozyma genus.

SEQUENCE SUBMISSION

The present application includes a Sequence Listing in electronic format. The Sequence Listing is entitled 2577230US2RevSequenceListing.txt, was created on 6 Feb. 2018 and is 40 kb in size. The information in the electronic format of the Sequence Listing is part of the present application and is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to the field of fungal biotechnology, more particularly to strong gene expression systems in species in the Pucciniomycotina and Ustilaginomycotina subphyla.

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.

The Pucciniomycotina is a subphylum of fungi in the phylum of Basidiomycota (Kirk et al., 2008). It holds many species that have important industrial applications. For example, a number of species in theRhodosporidiumandSporidiobolusgenera, such asRhodosporidium toruloides(also known asRhodotorula gracilis, Rhodosporidium glutinis, Rhodotorula glutinis, Torula koishikawensisandTorula rubescens) andSporobolomyces salmonicolor, are oil-rich single-cell yeasts capable of high density fermentation (Hu et al., 2009; Meng et al., 2009). These species hold great potential as a host for the production of long chain hydrocarbons, such as triacylglycerol (TAG, or fat), fatty acid esters (biodiesel), fatty alcohols, alcohols, lactones, terpenoids and vitamins (Wu et al., 2010a; Wu et al., 2010b; Zhao et al., 2010a; Zhao et al., 2010b). In another example, species in Ustilaginomycotina subphylum, in particular,UstilagoandPseudozymagenera, are known to produce glycolipids, which may function as a surfactant or fungicide (Hewald et al., 2005; Teichmann et al., 2010).

Promoters that are able to drive strong gene expression, either constitutively or inducibly, are critical for the development of biotechnological applications of a microorganism. WO 2012/169969, incorporated by reference herein in its entirety, describes several polynucleotide sequences derived from the upstream region of glyceraldehyde phosphate dehydrogenase gene (GPD1), translation initiation factor gene (TEF1), and putative stearoyl-CoA-delta 9-desaturase gene (FAD1) of selected fungal species that are able to function as a strong promoter of gene expression in Pucciniomycotina and Ustilaginomycotina subphyla. As repeated use of the identical or highly homologous promoter risks genome instability, epigenetic and genetic modification of chromatin resulted from repeat induced point mutation (RIP) or RNA silencing (Horns et al, 2012), an enlarged promoter pool is highly desirable for Pucciniomycotina and Ustilaginomycotina subphyla, wherein functionally verified promoters are scarce.

Promoters are DNA sequences located in the 5′ region adjacent to the transcriptional start site. It houses a combination of cis-acting DNA elements that act to interact with transcription factors by activating or repressing transcription of RNA polymerase. To date, genome shotgun sequences have been published forRhodotorula glutinisATCC 204091 (GenBank Accession: GL989638.1),Rhodosporidium toruloidesMTCC 457 (GenBank Accession: PRJNA112573),Rhodosporidium toruloidesNP11 (GenBank: ALAU00000000.1) and draft genome sequences have been published forRhodotorula graminisWP1 (http://genome.jgi-psf.org/Rhoba1_1/Rhoba1_1.home.html) andSporobolomyces roseus(http://genome.jgi-psf.org/Sporo1/Sporo1/Sporo1.home.html). RNA-Seq, proteomic and genome shotgun data released forRhodosporidium toruloidesNP11 (Zhu, Z., et al, 2012) are not able to define the sequence of functional promoters because the activity of a promoter is influenced by several factors, such as the location of 5′ and 3′ ends, posttranscriptional silencing, influence of intron, etc. The activity of a promoter in a heterologous host species is even more unpredictable.

SUMMARY OF THE INVENTION

The present invention relates to the field of fungal biotechnology, more particularly to strong gene expression systems in species in the Pucciniomycotina and Ustilaginomycotina subphyla.

In a first aspect, the present invention provides polynucleotide sequences that function as strong promoters of gene expression inRhodosporidium, Rhodotorula, Sporobolomyces, PseudozymaandUstilagogenera. These polynucleotide sequences are sometimes referred to herein as polynucleotide promoter sequences. In one embodiment, the polynucleotide promoter sequences comprises the sequence set forth in any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In another embodiment, the polynucleotide promoter sequences comprises the promoter sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, i.e., the sequence without the cloning sites. Each of the polynucleotide promoter sequences contains at least one GAGGAG sequence motif, which functions to enhance gene expression in said fungal species. Each of polynucleotide sequences is effective in performing strong gene expression inRhodosporidium, Rhodotorula, Sporobolomyces, PseudozymaandUstilagogenera. In addition, operable fragments of these polynucleotide promoter sequences can be isolated using convention promoter screening assays and can be screened for efficient selection of transformed fungal cells using the techniques described herein. In one embodiment, an operable fragment, also termed a promoter portion herein, is about 400 base pairs up to about 1100 base pairs in length starting from the −1 position from the ATG codon. As used herein “up to” refers to the length of the promoter portion of the promoters set forth in the disclosed SEQ ID NOs. Thus, “up to” refers to the maximal length of the promoter sequence if less than 1100 nucleotides of the promoters of the disclosed SEQ ID NOs.

In a second aspect, the present invention provides a DNA construct comprising the polynucleotide promoter sequences described herein, an operably linked polypeptide encoding sequence and an operably linked RNA transcriptional terminator sequence. Any eukaryotic transcriptional terminator, well known to the skilled artisan, may be used. Such a DNA construct allows strong expression of the polypeptide in a fungal species in which the genome is biased in C and G. Of particular relevance are species selected from the Pucciniomycotina and Ustilaginomycotina subphyla. The species of particular relevance are those in theRhodosporidium, Rhodotorula, Sporobolomyces, UstilagoandPseudozymagenera, in which reside a number of species with great potential for the bioconversion of renewable resources into high-value products, such as triglyceride, biodiesel, fatty alcohol, vitamins, lactone, terpenoids and biosurfactants.

DETAILED DESCRIPTION OF THE INVENTION

The term “operably linked” or “operatively linked” is defined herein as a configuration in which a regulatory or control sequence is appropriately placed at a position relative to the nucleotide sequence of the nucleic acid construct such that the control sequence directs the expression of a polynucleotide of the present invention. Regulatory or control sequences may be positioned on the 5′ side of the nucleotide sequence or on the 3′ side of the nucleotide sequence as is well known in the art.

The term “strong expression” as used herein means expression of a marker protein or mRNA to a detectable level using detection methods known, for example, florescence for GFP, activity assay for GUS and lacZ genes.

The present invention relates to the field of fungal biotechnology, more particularly to strong gene expression systems in species in the Pucciniomycotina and Ustilaginomycotina subphyla.

In a first aspect, the present invention provides polynucleotide sequences that function as strong promoters of gene expression inRhodosporidium, Rhodotorula, Sporobolomyces, PseudozymaandUstilagogenera. These polynucleotide sequences are sometimes referred to herein as polynucleotide promoter sequences. In one embodiment, the polynucleotide promoter sequences comprises the sequence set forth in any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In another embodiment, the polynucleotide promoter sequences comprises the promoter sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, i.e., the sequence without the cloning sites. Each of the polynucleotide promoter sequences contains at least one GAGGAG sequence motif, which functions to enhance gene expression in said fungal species. Each of polynucleotide sequences is effective in performing strong gene expression inRhodosporidium, Rhodotorula, Sporobolomyces, PseudozymaandUstilagogenera. In addition, operable fragments of these polynucleotide promoter sequences can be isolated using convention promoter screening assays and can be screened for efficient selection of transformed fungal cells using the techniques described herein. In one embodiment, an operable fragment, also termed a promoter portion herein, is about 400 base pairs up to about 1100 base pairs in length starting from the −1 position from the ATG codon. As used herein “up to” refers to the length of the promoter portion of the promoters set forth in the disclosed SEQ ID NOs. Thus, “up to” refers to the maximal length of the promoter sequence if less than 1100 nucleotides of the promoters of the disclosed SEQ ID NOs.

In a second aspect, the present invention provides a DNA construct comprising the polynucleotide promoter sequences described herein, an operably linked polypeptide encoding sequence and an operably linked RNA transcriptional terminator sequence. Any eukaryotic transcriptional terminator, well known to the skilled artisan, may be used. Such a DNA construct allows strong expression of the polypeptide in a fungal species in which the genome is biased in C and G. Of particular relevance are species selected from the Pucciniomycotina and Ustilaginomycotina subphyla. The species of particular relevance are those in theRhodosporidium, Rhodotorula, Sporobolomyces, UstilagoandPseudozymagenera, in which reside a number of species with great potential for the bioconversion of renewable resources into high-value products, such as triglyceride, biodiesel, fatty alcohol, vitamins, lactone, terpenoids and biosurfactants.

Nucleic acid hybridization, a technique well known to those of skill in the art of DNA manipulation, can be used to identify other suitable polynucleotides. In accordance with the invention other suitable promoters for use may be obtained by the identification of polynucleotides that selectively hybridize to the promoters described above by hybridization under low stringency conditions, moderate stringency conditions, or high stringency conditions. Selectively hybridizing sequences typically have at least 50% sequence identity, preferably at least 70%, 80% or 90% sequence identity, and most preferably 95%, 98% or 99% sequence identity with each other.

Database searches and homology searches of genome and nucleotide databases identify similar DNA or RNA molecules based on the alignment of nucleotides using algorithms or computer programs and these techniques well known to those of skill in the art. In accordance with the invention other suitable polynucleotides for use may be obtained by the in silico identification of polynucleotides for regulatory sequences with at least 50% sequence identity, preferably at least 70%, 80% or 90% sequence identity, and most preferably 95%, 98% or 99% sequence identity with each other.

The invention provides a polynucleotide promoter sequence selected from SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or the promoter sequence thereof, i.e., the sequence without the cloning sites. In one embodiment, a polynucleotide promoter sequence is provided which has at least 60% identity with any one of these polynucleotide promoter sequences. In another embodiment, a polynucleotide promoter sequence is provided which has at least 70% identity with any one of these polynucleotide promoter sequences. In an additional embodiment, a polynucleotide promoter sequence is provided which has at least 80% identity with any one of these polynucleotide promoter sequences. In a further embodiment, a polynucleotide promoter sequence is provided which has at least 90% identity with any one of these polynucleotide promoter sequences. In another embodiment, a polynucleotide promoter sequence is provided which has at least 95% identity with any one of these polynucleotide promoter sequences. In another embodiment, a polynucleotide promoter sequence is provided which has at least 98% identity with any one of these polynucleotide promoter sequences. In one embodiment, a promoter sequence herein, is about 400 base pairs up to about 1100 base pairs in length starting from the −1 position from the ATG codon. As used herein “up to” refers to the length of the promoter portion of the promoters set forth in the disclosed SEQ ID NOs. Thus, “up to” refers to the maximal length of the promoter sequence if less than 1100 nucleotides of the promoters of the disclosed SEQ ID NOs.

The invention provides a polynucleotide construct comprising an isolated promoter described herein, such as one selected from SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, or promoter portion thereof, operatively linked to a polypeptide-encoding sequence which is operatively linked to a transcriptional terminator. In one embodiment, an operable fragment, also termed a promoter portion herein, is about 400 base pairs up to about 1100 base pairs in length starting from the −1 position from the ATG codon. As used herein “up to” refers to the length of the promoter portion of the promoters set forth in the disclosed SEQ ID NOs. Thus, “up to” refers to the maximal length of the promoter sequence if less than 1100 nucleotides of the promoters of the disclosed SEQ ID NOs. In one embodiment, the polynucleotide construct enables efficient expression of a polypeptide in a fungal species selected from Pucciniomycotina and Ustilaginomycotina subphyla. The fungal species is preferably one selected fromRhodosporidium, Rhodoturula, Ustilago, Pseudozyma, orSporobolomycesgenus, the genome of which contains at least 50% C and G, preferably more than 60% C and G.

In one embodiment, the polynucleotide construct is inserted in a T-DNA vector, a shuttle vector, or in a fungal chromosome, wherein the polypeptide-encoding sequence contains at least 50% CG, preferably 60% CG and most preferably more than 80% CG.

In one embodiment, any transcriptional terminator operable in species of the fungi can be used. Terminators are typically located downstream (3′) of the gene, after the stop codon (TGA, TAG or TAA). Terminators play an important role in the processing and stability of RNA as well as in translation. Most, but not all terminators, contain a polyadenylation sequence or cleavage site. Examples of specific polyadenylation sequences are AAUAAA or AAUAAU. These sequences are known as the near upstream elements (NUEs) (Nagaya et al., 2010). NUEs usually reside approximately 30 bp away from a GU-rich region (Mogen et al., 1990; Mogen et al., 1992; Rothnie et al. 1994), known as far upstream elements (FUEs). The FUEs enhance processing at the polyadenylation sequence or cleavage site, which is usually a CA or UA in a U-rich region (Bassett, 2007). Within the terminator, elements exist that increase the stability of the transcribed RNA (Ohme-Takagi et al., 1993; Newman et al., 1993; Gutierrez et atl., 1999) and may also control gene expression (Ingelbrecht, 1989; An et al., 1989).

Nucleic acid hybridization, a technique well known to those of skill in the art of DNA manipulation, can be used to identify other suitable terminators. In accordance with the invention other suitable promoters for use may be obtained by the identification of terminators that selectively hybridize to the promoters described above by hybridization under low stringency conditions, moderate stringency conditions, or high stringency conditions. Selectively hybridizing sequences typically have at least 50% sequence identity, preferably at least 70%, 80% or 90% sequence identity, and most preferably 95%, 98% or 99% sequence identity with each other.

Database searches and homology searches of genome and nucleotide databases identify similar DNA or RNA molecules based on the alignment of nucleotides using algorithms or computer programs and these techniques well known to those of skill in the art. In accordance with the invention other suitable terminators for use may be obtained by the in silico identification of terminators for regulatory sequences with at least 50% sequence identity, preferably at least 70%, 80% or 90% sequence identity, and most preferably 95%, 98% or 99% sequence identity with each other.

A DNA of interest can be added to the polynucleotide construct. The DNA of interest is operatively linked to promoter and a terminator. Any promoter and terminator operable in species of the Pucciniomycotina and Ustilaginomycotina subphyla can be used. In some embodiments, the DNA of interest may be used to insert or modify metabolic pathways, such as fatty acid biosynthesis, lipid biosynthesis, triglyceride biosynthesis, and the like. The DNA of interest may be inserted into the genome of the fungal cells to enhance the bioconversion of renewable resources into high-value products, such as triglycerides, biodiesel, fatty alcohol, vitamins, biosurfactants, lactone, terpenoid and the like.

A polynucleotide construct may be introduced directly into the genomic DNA of the fungal cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the polynucleotide constructs can be introduced directly to fungal tissue using ballistic methods, such as DNA particle bombardment. Alternatively, the polynucleotide constructs may be combined with suitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefacienshost vector. The virulence functions of theAgrobacterium tumefacienshost will direct the insertion of the construct into the fungal cell DNA when the cell is infected by the bacteria. Thus, any method, which provides for effective transformation/transfection may be employed. See, for example, U.S. Pat. Nos. 7,241,937, 7,273,966 and 7,291,765 and U.S. Patent Application Publication Nos. 2007/0231905 and 2008/0010704 and references cited therein. See also, International Published Application Nos. WO 2005/103271 and WO 2008/094127 and references cited therein.

The transformed fungi are transferred to standard growing media (e.g., solid or liquid nutrient media, grain, vermiculite, compost, peat, wood, wood sawdust, straw, etc.) and grown or cultivated in a manner known to the skilled artisan.

After the polynucleotide is stably incorporated into transformed fungi, it can be transferred to other fungi by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

It may be useful to generate a number of individual transformed fungi with any recombinant construct in order to recover fungi free from any positional effects. It may also be preferable to select fungi that contain more than one copy of the introduced polynucleotide construct such that high levels of expression of the recombinant molecule are obtained.

It may be desirable to produce fungal lines that are homozygous for a particular gene if possible in the particular species. In some species this is accomplished by the use monosporous cultures. By using these techniques, it is possible to produce a haploid line that carries the inserted gene and then to double the chromosome number either spontaneously or by the use of colchicine. This gives rise to a fungus that is homozygous for the inserted gene, which can be easily assayed for if the inserted gene carries with it a suitable selection marker gene for detection of fungi carrying that gene. Alternatively, fungi may be self-fertilized, leading to the production of a mixture of spores that consists of, in the simplest case, three types, homozygous (25%), heterozygous (50%) and null (25%) for the inserted gene. Although it is relatively easy to score null fungi from those that contain the gene, it is possible in practice to score the homozygous from heterozygous fungi by Southern blot analysis in which careful attention is paid to the loading of exactly equivalent amounts of DNA from the mixed population, and scoring heterozygotes by the intensity of the signal from a probe specific for the inserted gene. It is advisable to verify the results of the Southern blot analysis by allowing each independent transformant to self-fertilize, since additional evidence for homozygosity can be obtained by the simple fact that if the fungi was homozygous for the inserted gene, all of the subsequent fungal lines from the selfed individual will contain the gene, while if the fungus was heterozygous for the gene, the generation grown from the selfed seed will contain null fungal lines. Therefore, with simple selfing one can select homozygous fungal lines that can also be confirmed by Southern blot analysis.

Creation of homozygous parental lines makes possible the production of hybrid fungus and spores that will contain a modified protein component. Transgenic homozygous parental lines are maintained with each parent containing either the first or second recombinant DNA sequence operably linked to a promoter. Also incorporated in this scheme are the advantages of growing a hybrid crop, including the combining of more valuable traits and hybrid vigor.

EXAMPLES

The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

Culture of Microbial Strains and Basic Molecular Methods

Genomic DNA was extracted based on the method described forU. maydis(Ji et al., 2010) with some modifications. Briefly, the cell culture at exponential phase was collected and washed with 1 M sorbitol. The cells were resuspended in 0.1 ml of SCS buffer (1 M sorbitol, 20 mM sodium citrate, pH 5.8) and supplemented with glass beads (1 mm in diameter, Sigma-Aldrich, USA). Cells lysis made by vortexing and genomic DNA was isolated after phenol/chloroform extraction and ethanol precipitated. The extracted DNA was quantified with NanoDrop® ND-1000 Spectrophotometer (Nanodrop Technologies, USA) and DNA quality analyzed by agarose gel electrophoresis.

Cloning of Promoters

Based on the published EST sequence abundance in various media (Ho et al, 2007), we selected a number of genes (Table 1) as potential source of strong promoters forRhodosporidiumandRhodotorula. Other candidate genes include those encoding proteins in the fatty acid biosynthesis inRhodosporidiumandRhodotorula, e.g., acetyl-CoA synthase (ACC1), acyl-CoA carrier protein (ACP1), pyruvate decarboxylase (PDC1) and nitrate regulated gene (NAR1).Ustilago maydisCDS sequences were searched against theRhodosporidiumandRhodotorulagenome database.

To define the 3′ end of the promoters, 5′ RACE were performed using BD SMARTer™ RACE cDNA Amplification Kit (Clontech, California, USA) according to the manufacturer's instruction. Promoter DNA fragments were obtained by PCR using a 3′ end primer that is designed at the first ATG in the 5′ untranslated regions, usually with an overlapping NcoI (CCATGG) or BspHI (TCATGA) site at the ATG codon. BamHI is used if the DNA sequence contains both NcoI and BspHI sites. 5′ Primers were designed 1-2 kb from the ATG. The primers used are listed in Table 2. The PCR fragments were digested with corresponding enzymes, cloned in pPN006 or pRH2031 (FIG. 1), which is a T-DNA vector containing the RtGPD1::RtGFP:nos cassette (Liu et al, 2012).

Promoter Activity inRhodosporidiumin Lipid Production Medium

The promoter GFP reporter constructs were transformed toRhodosporidium toruloidesATCC 10657 by the ATMT method. The transformed colonies (>100) were pooled, cultured in YPD medium with 150 μg/ml hygromycin B and 300 μg/ml cefataxome and diluted to about 0.1 OD600 in lipid production medium [10 mM K2HPO4-KH2PO4, (pH6.13), 4 g/L yeast extract, 0.3 g/L urea, 0.1 g/L Na2SO4, 10 mg/L each of tyrosine, valine and vitamin B (B1+B6), 8% glucose] with no antibiotics added. Strains were cultured at 28° C. with shaking (280 rpm) 2 days and then dilute to about 0.1 OD600 for 24 hours and the cultures were adjusted to 0.6 OD600 units before subjecting to florescence measurement in a Tecan M200 reader using 476 nM as excitation wavelength, and 509 nM as emission wavelength; gain value 100. The florescence intensity were normalized to OD600 and subtracted against non-transformed cell cultures under the same conditions. Rg3 TAL1 is weak while Rg2ACC1 showed no obvious activity (Table 3). Transformants of the Rg2A CC1 reporter inRhodosporidium glutinisATCC 90781 showed no GFP florescence was cultured a nitrogen-limited medium (glucose 70 g/l, yeast extract 0.75 g/l, (NH4)2SO4 0.1 g/l, KH2PO4 1.0 g/l, MgSO4.7H2O 1.5 g/l, pH 5.6) for 24 hr either (not shown). Promoter RtGPD1 is set forth in SEQ ID N:38.

TABLE 3Relative GFP Fluorescence of Selected Promoters inRhodosporidium toruloidesPromoterRg2TEF1Rg3TEF1Rg3TAL1Rg3S27Rg3PPIRg2ENO1Rg2ACC1RtGPD1Fluorescence1836168331656638301100reading

A selected set of promoter GFP reporter constructs were transformed by the ATMT method toRhodosporidium toruloidesATCC 10657,Ustilago maydisL8,Pseudozyma aphidisATCC32657 andSporobotomyces roseusFGSC 10293. The transformed colonies (>100) were pooled, cultured in YPD medium with 150 μg/ml (or 300 μg/ml forPseudozyma aphidis) hygromycin B and 300 μg/ml cefataxome for 2 days at 28° C. and diluted to about 0.1 OD600 in YNB Medium and also in YNB N-Medium (both medium with 5% gluocose.) and continued culture for 1-3 days with shaking. OD600 and GFP florescence were measured with Tecan infinite200. The GFP fluorescence intensity (normalized against the OD600) is listed in Tables 4-11. Promoters Umgpd1, RtGPD1, Rg3GPD1, Rg2FAD1 and SrGPD1 are set forth in SEQ IN NOs:37, 38, 39, 40 and 41, respectively. The isolation of these promoters is described in WO 2012/169969.

Identification of Critical Elements for Strong Promoters

Promoter sequences of Rg3TEF1, Rg3S27, Rg2ACP1, Rg2ENO1, Rg2PDC1, Rg3PDC1 and Rg2FAD1 (stearoyl-CoA delta-9 desaturase) were subjected to promoter motif scanning using the Gibbs Motif Sampler at http://ccmbweb.ccv.brown.edu/cgi-bin/gibbs.12.pl?data13type=DNA&layout=advancedprgm&restore=var/www/cgi-bin/euk.def.txt.

A conserved motif sharing the GAGGAG core sequence were found in each promoter. Noticeably, Rg2FAD1 promoter, which is among the strongest promoters contains the largest number of the motif. (FIG. 2).

Nested Deletion of Rg2FAD1 and Rg2ENO1 Promoters

The full length Rg2ENO1 and Rg2FAD1 (stearoyl-CoA delta-9 desaturase gene) promoter GFP reporter constructs in pRH2031 were modified to have serially shortened promoters. This was done replacing the promoter with PCR fragments about 300, 500, 1000 and 1500 bp version of the promoter. All 5′ primers included a SpeI cutting site while the 3′ primer contains a NcoI cutting site. Constructs were transformed by the ATMT method toRhodosporidium toruloidesATCC 10657. The transformed colonies (>500) were pooled, cultured in YNB medium with 150 μg/ml hygromycin B and 300 μg/ml cefataxome for 2 days at 28° C. and diluted to about 0.1 OD600in YNB Medium and also in YNB N−Medium (both medium with 5% glucose.) and continued culture for 24 hours with shaking. The cultures reached OD6000.2 units in YNB N+ and YNB N−media. GFP florescence were measured with Tecan infinite M200. Gain parameter is consistently set at 85; Excitation and Emission wavelength are 476, 509. The GFP fluorescence intensity (normalized against the OD600) is listed inFIG. 3, which shows that minimal length of the ENO1 promoter for the optimal expression of reporter gene is approximately 320 to 520 bp, whereas the FAD1 promoter requires approximately 570 to 1120 bp.

Nested Deletion of 519 bp Rg2ENO1 Promoter

Primers were designed at various locations in the 519 bp Rg2ENO1 promoter sequence, which were used for PCR in combination with the reverse primer targeting the 3′ end of the promoter (FIG. 4A). All 5′ primers included a SpeI cutting site while the 3′ primer contains a NcoI cutting site. The lengths of the PCR products (excluding the extra linker sequence at 5′ end and the ATG codon at the 3′ site) are summarized inFIG. 4B. The PCR fragments were individually digested with SpeI and NcoI and cloned in pRH2031-Rg2ENO1-RtGFP at the same sites, replacing the full-length ENO1 promoter. Constructs were transformed by the ATMT method toRhodotorula glutinisATCC 90781, which is the diploid parent ofRhodosporidium toruloidesATCC 10657 and ATCC 10788. The transformed colonies (>500) were pooled, cultured in YNB medium with 150 μg/ml hygromycin B and 300 μg/ml cefataxome for 2 days at 28° C. and diluted to about 0.1 OD600in YNB Medium and also in YNB N−Medium (both medium with 5% glucose.) and continued culture for 12 hours with shaking. The cultures reached OD6000.5˜0.7 in YNB and YNB N−meda. GFP florescence was measured with Tecan infinite M200. Gain parameter is consistently set at 85; Excitation and Emission wavelength are 476, 509. The GFP fluorescence intensity (normalized against the OD600) is listed inFIGS. 5A and 5B. The promoter showed similar trends in the two media tested. The biggest drop in activity was seen promoter M6 and M7. Another significant drop was observed between M3 and M6, where several GAGGAG-related motifs can be found (FIG. 4A).

The term “efficient expression” refers to expression of a reporter protein to a level that is detectable for fluorometry, photomicrospy or phenotypic selection of transformants by antibiotics, such as hygromycin.

BIBLIOGRAPHY