Targeted integrative transformation of Candida tropicalis by electroporation

Summary A method for integrative transformation of the diploid yeast Candida tropicalis by electroporation has been developed. By linearizing the transforming plasmid DNA containing the URA3 gene prior to electroporation of recipient cells, its integration was targeted to a specific locus in the genome, resulting in single or multiple tandem integrations. The optimal electroporation conditions for this yeast were established and include an electric pulse of 2.25 kV/cm for a duration of 50 ms. Using these conditions, Ura⁺ transformants were readily obtained at a high frequency (45 transformants/g DNA) as the result of targeted integration of the URA3 gene containing plasmid DNA at the chromosomal ura3 locus. The number of transformants resulting from this procedure is comparable to that achieved with a recently reported spheroplast transformation procedure for C. tropicalis; in addition, it offers the advantages of being simple, rapid and reproducible.     

Homologous gene targeting of a carotenoids biosynthetic gene in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation

Objectives

To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method.

Results

The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11.

Conclusions

Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.

     

Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous <Emphasis Type="Italic">GFP</Emphasis> genes in <Emphasis Type="Italic">Candida tropicalis</Emphasis>

The diploid yeast Candida tropicalis, which can utilize n-alkane as a carbon and energy source, is an attractive strain for both physiological studies and practical applications. However, it presents some characteristics, such as rare codon usage, difficulty in sequential gene disruption, and inefficiency in foreign gene expression, that hamper strain improvement through genetic engineering. In this work, we present a simple and effective method for sequential gene disruption in C. tropicalis based on the use of an auxotrophic mutant host defective in orotidine monophosphate decarboxylase (URA3). The disruption cassette, which consists of a functional yeast URA3 gene flanked by a 0.3 kb gene disruption auxiliary sequence (gda) direct repeat derived from downstream or upstream of the URA3 gene and of homologous arms of the target gene, was constructed and introduced into the yeast genome by integrative transformation. Stable integrants were isolated by selection for Ura⁺ and identified by PCR and sequencing. The important feature of this construct, which makes it very attractive, is that recombination between the flanking direct gda repeats occurs at a high frequency (10^?8) during mitosis. After excision of the URA3 marker, only one copy of the gda sequence remains at the recombinant locus. Thus, the resulting ura3 strain can be used again to disrupt a second allelic gene in a similar manner. In addition to this effective sequential gene disruption method, a codon-optimized green fluorescent protein-encoding gene (GFP) was functionally expressed in C. tropicalis. Thus, we propose a simple and reliable method to improve C. tropicalis by genetic manipulation.     

Integrative Transformation System for the Metabolic Engineering of the Sphingoid Base-Producing Yeast Pichia ciferrii

We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/μg of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.     

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.     

Marker-disruptive gene integration and URA3 recycling for multiple gene manipulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The introduction of several kinds of genes into the yeast chromosome is a powerful tool in many fields from fundamental study to industrial application. Here, we describe a general strategy for one-step gene integration and a marker recycling method. Forty base pairs of a short sequence derived from a region adjacent to the HIS3 locus were placed between cell surface displaying β-glucosidase (BGL) and URA3 marker genes. HIS3 deletion and BGL–URA3 fragment integration were achieved via a PCR fragment consisting of the BGL–URA3 fragment attached to homology sequences flanked by the HIS3 targeting locus. The obtained his3::URA3 disruptants were plated on a 5-FOA plate to select for the URA3 deletion due to repeated sequences at both sides of URA3 gene. In all selected colonies, BGL genes were integrated at the targeted HIS3 locus and URA3 was completely deleted. In addition, introduced BGL was efficiently expressed, and the transformants fermented cellobiose to ethanol effectively. As our strategy creates next transformation markers continuously together with gene integration, this method can serve as a simple and powerful tool for multiple genetic manipulations in yeast engineering.     

Efficient Transformation of the Astaxanthin-Producing Yeast Phaffia Rhodozyma

An efficient transformation system for the astaxanthin-producing yeast Phaffia rhodozyma was developed based on electroporation that routinely yields approximately 1000 transformants per g of plasmid DNA. The high transformation efficiency depends on vector integration in the ribosomal DNA (rDNA) and the presence of the homologous glycolytic glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and terminator to drive the expression of the transposon Tn5 encoded kanamycin resistance gene (Km^R) as a selective marker. Using this system stable transformants were obtained, carrying multiple plasmid copies. Plasmid copy number could be markedly increased by deletion of the gpd terminator from the transforming plasmid.     

Rare Homologous Gene Targeting in Histoplasma capsulatum: Disruption of the URA5Hc Gene by Allelic Replacement

URA5 genes encode orotidine-5′-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the Histoplasma capsulatum URA5 gene (URA5_Hc) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized ura5 strains of H. capsulatum. We attempted to target the genomic URA5 locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in H. capsulatum and has been challenging in some other pathogenic fungi. Several strategies commonly used in Saccharomyces cerevisiae and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for URA5 gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (hph, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Δura5_Hc::hph plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in H. capsulatum, or in any dimorphic systemic fungal pathogen of humans.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis

Two methods of multicopy integrant selection in the methylotrophic yeast Hansenula polymorpha based on the use of heterologous yeast auxotrophic genes have been used to isolate effective overproducers of hepatitis B surface antigen (HBsAg). One selection marker was described earlier for this yeast, the Saccharomyces cerevisiae URA3 gene, whereas the second selection marker was developed by us, the Pichia pastoris ADE1 gene with shortened native promoter. Sequential use of both selection markers produced stable transformants containing up to 30 integration cassettes with HBsAg gene. Deletion of PEX3 gene coding for peroxine involved in the early step of peroxisome formation substantially increased the production of HBsAg in glucose medium as compared to the parental strain. Maximal production of HBsAg in Δpex3 strain was nearly 8–9 % of the total cell protein.     

One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica

Yarrowia lipolytica is an unconventional yeast, and is generally recognized as safe (GRAS). It provides a versatile fermentation platform that is used commercially to produce many added-value products. Here we report a multiple fragment assembly method that allows one-step integration of an entire β-carotene biosynthesis pathway (~11 kb, consisting of four genes) via in vivo homologous recombination into the rDNA locus of the Y. lipolytica chromosome. The highest efficiency was 21 %, and the highest production of β-carotene was 2.2 ± 0.3 mg per g dry cell weight. The total procedure was completed in less than one week, as compared to a previously reported sequential gene integration method that required n weeks for n genes. This time-saving method will facilitate synthetic biology, metabolic engineering and functional genomics studies of Y. lipolytica.     

Development of an integrative DNA transformation system for the yeast Hansenula anomala

A host-vector system for the yeast Hansenula anomala was developed. The system was based on an auxotrophic mutant host of H. anomala which was defective in orotidine-5′-phosphate decarboxylase (ODCase) activity. The H. anomala ODCase-negative mutant strains (ura3 strains) were isolated based on 5-fluoroorotic acid (5-FOA) resistance. A plasmid vector containing the H. anomala URA3 gene was used for transformation. Using this plasmid, all of the H. anomala ura3 strains tested could be transformed to Ura⁺ phenotypes. In all of Ura⁺ transformants, the introduced plasmid was integrated into the chromosomal URA3 locus by homologous recombination. The Ura⁺ phenotype of the transformants was stably maintained after nonselective growth.     

Disruption of the gene encoding restriction endonuclease <Emphasis Type="Italic">Sua</Emphasis>I and development of a host–vector system for the thermoacidophilic archaeon <Emphasis Type="Italic">Sulfolobus acidocaldarius</Emphasis>

Sulfolobus acidocaldarius is a useful model organism for the genetic study of thermophilic archaea due to its ease of cultivation. Here we describe the development of a host–vector system for S. acidocaldarius consisting of SuaI restriction system-deficient strain SK-1 and shuttle vector pSAV2. The new host strain SK-1 was constructed by pop-out recombination based on the pyrE marker gene. Plasmid pSAV2 was constructed from the S. islandicus native plasmid pRN1, in which selectable markers and functional genes were inserted in suitable locations and orientations followed by the deletion of non-essential open reading frames. SK-1 allowed direct transformation without N⁴-methylation at SuaI restriction sites, so unmethylated vector pSAV2 could be introduced directly into SK-1 by electroporation. The transformants were selected by pyrEF complementation on xyrose–tryptone solid medium without prior liquid culturing. The transformation efficiency was approximately 1.0 × 10³/μg DNA. After replication in S. acidocaldarius, pSAV2 was successfully recovered from transformant cultures by the standard alkaline lysis method. Plasmid yield was approximately 40–50 ng/ml from late-log through stationary phase cultures. In addition, pSAV2 was maintained stably and at relatively high copy number in S. acidocaldarius.     

Stable yeast transformation with chimeric plasmids using a 2 micron-circular DNA-less strain as a recipient.

Summary By using two chimeric plasmids containing yeast URA3 gene as a selection marker and 2 m yeast DNA linked to the bacterial plasmid pCR1, a yeast strain devoid of any 2 m DNA sequence was transformed. Recovery in E. coli of plasmids from yeast transformants showed that the 2 m-less strain was able to maintain the chimeric plasmids as autonomous replicons, with very infrequent plasmid recombination. Hybridization experiments gave no evidence for integration of the URA3 DNA sequence in the chromosomal DNA. The transformed clones showed a high stability of the ura⁺ character during vegetative multiplication, even in the absence of selective pressure. The specific activity of orotidine 5 monophosphate decarboxylase (coded by the URA3 gene) was 5 to 10 fold higher than in the wild type.These features should offer new possibilities for cloning with yeast.     

Genetic Transformation System for the Fungal Soybean Pathogen Cercospora kikuchii

An altered β-tubulin gene that confers resistance to the fungicide benomyl was isolated from a genomic library of a UV-induced mutant of Cercospora kikuchii and used as a selectable marker for transformation. The level of benomyl resistance conferred to the transformants was at least 150-fold greater than the intrinsic resistance of the C. kikuchii recipient protoplasts. In the majority of cases, the tubulin fragment was integrated at the native β-tubulin locus, apparently by gene replacement or gene conversion. The frequency of transformation ranged from 0.2 to 6 transformants per μg of DNA, depending on the recipient strain. Transformation with linearized plasmid resulted in a higher frequency, without changing the type of integration event. Transformants were phenotypically stable after eight consecutive transfers on medium without benomyl. This is the first report of a genetic transformation system for a Cercospora species.     

An efficient gene disruption method using a positive–negative split-selection marker and <Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-mediated transformation for <Emphasis Type="Italic">Nomuraea rileyi</Emphasis>

Targeted gene disruption via Agrobacterium tumefaciens-mediated transformation (ATMT) and homologous recombination is the most common method used to identify and investigate the functions of genes in fungi. However, the gene disruption efficiency of this method is low due to ectopic integration. In this study, a high-efficiency gene disruption strategy based on ATMT and the split-marker method was developed for use in Nomuraea rileyi. The β-glucuronidase (gus) gene was used as a negative selection marker to facilitate the screening of putative transformants. We assessed the efficacy of this gene disruption method using the NrCat1, NrCat4, and NrPex16 genes and found that the targeting efficiency was between 36.2 and 60.7%, whereas the targeting efficiency using linear cassettes was only 1.0–4.2%. The efficiency of negative selection assays was between 64.1 and 82.3%. Randomly selected deletion mutants exhibited a single copy of the hph cassette. Therefore, high-throughput gene disruption could be possible using the split-marker method and the majority of ectopic integration transformants can be eliminated using negative selection markers. This study provides a platform to study the function of genes in N. rileyi.     

Size Of Gene Specific Inverted Repeat - Dependent Gene Deletion In Saccharomyces cerevisiae

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.