Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus
<p>Construction of the plasmid series pRS50 (integrative plasmids) with a detailed list of intermediate cloning steps and relevant restriction sites. ble = original ble ORF from Klebsiella pneumoniae, EgTEFpromoter = promoter of the Eremothecium gossypii TEF gene, EgTEFterminator = terminator of the E. gossypii TEF gene, oBle = codon-optimized ble ORF encoding for resistance to phleomycin, oHyg = codon-optimized ORF encoding for resistance to hygromycin B, oKan = codon-optimized ORF encoding for resistance to G418, oNrs = codon-optimized ORF encoding for resistance to clonNAT, bla = resistance to ampicillin, ori = replication origin from the <span class="html-italic">E. coli</span> plasmid pBR322, lacZα = gene encoding α-peptide required for the blue-white screening.</p> "> Figure 2
<p>Construction of the plasmid series pRS52, pRS53, pRS54, pRS55, and pRS56 (replicative plasmids, each carrying one yeast replication origin) with a detailed list of intermediate cloning steps and relevant restriction sites. 2µ ori, panARS, SsARS2, MgALS123, BbCEN2 = yeast replication origins, ScLEU2 = LEU2 gene from <span class="html-italic">Saccharomyces cerevisiae</span>, rop = gene encoding Rop protein in <span class="html-italic">E. coli</span>. Other labels are identical to those in <a href="#jof-08-00531-f001" class="html-fig">Figure 1</a>.</p> "> Figure 3
<p>Schematic representation of integrative and replicative yeast plasmids constructed in this work and their names and main features. The pRS40 backbone carries sequences for selection and maintenance in <span class="html-italic">E. coli</span> (ori, bla) and other common vector features (<span class="html-italic">LacZ</span><span class="html-italic">α</span>, <span class="html-italic">f1 ori</span>), as well as TEF promoter (P<span class="html-italic"><sub>Eg</sub></span><sub>TEF</sub>) and terminator (T) from <span class="html-italic">Eremothecium gossypiii</span> for expression of antibiotic selectable markers in yeasts. Codon usage optimized markers conferring resistances to geneticin G418 (Kan<sup>R</sup>), hygromycin B (Hyg<sup>R</sup>), cloNAT (Nar<sup>R</sup>) or phleomycin (Phl<sup>R</sup>) were cloned under the regulation of the TEF promoter thus creating four different yeast integrative plasmids. Additionally, in each of these plasmids, the following origins of the replications were cloned: 2μ form <span class="html-italic">Saccharomycer cerevisiae</span>, panARS form <span class="html-italic">Kluyveromyces lactis</span>, <span class="html-italic">Ss</span>ARS2 from <span class="html-italic">Scheffersomyces stipitis</span>, <span class="html-italic">Mg</span>ALS123 form <span class="html-italic">Merozyma guilliermondii</span> or <span class="html-italic">Bb</span>CEN2 from <span class="html-italic">Brettanomyces bruxellensis</span>, thus creating a set of 20 replicative yeast plasmids.</p> "> Figure 4
<p>Typical results of molecular analysis by Southern blotting of undigested (<b>A</b>) and digested (<b>B</b>) DNA isolated form transformants of <span class="html-italic">S. polymorphus var. africanus</span>.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Growth Conditions
2.2. Yeast Strains, Plasmids and Oligonucleotides
2.3. Gene Optimisation, Synthesis and Testing
2.4. Plasmid Construction
2.4.1. Construction of Integrative Plasmids
2.4.2. Construction of Replicative Plasmids
2.5. Determination of Yeast Antibiotic Resistance
2.6. Yeast Transformation and Molecular Analysis by Southern Blotting
3. Results and Discussion
3.1. Plasmid Set for Transformation of Yeasts Belonging to the Saccharomycotina Subphylum
3.1.1. Integrative Yeast Plasmids
3.1.2. Replicative Yeast Plasmids
3.2. Transformation of Yeasts Belonging to the Schwanniomyces Genus
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Geneticin G418 [μg/mL] | Hygromycin B [μg/mL] | cloNAT [μg/mL] | Phleomycin [μg/mL] | |
---|---|---|---|---|
S. cerevisiae | 200 | 300 | 100 | 10 |
K. lactis | 150 | 200 | 20 | 100 |
S. stipitis | selection not possible | 600 | 50 | 100 |
M. guilliermondii | selection not possible | 500 | 200 | 200 |
B. bruxellensis | 250 | 150 | 35 | 150 |
S. pseudopolymorpshus | selection not possible | 200 | 50 | Not determined |
S. polymorphus var. polymorphus | selection not possible | 200 | 50 | Not determined |
S. polymorphus var. africanus | selection not possible | 400 | 50 | Not determined |
Yeast Strains | Genotype | Reference |
---|---|---|
Saccharomyces cerevisiae BY 4742 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 | [11] |
Kluyveromyces lactis CBS 2359T | Type strain | Westerdijk Fungal Biodiversity Institute, The Netherlands |
Scheffersomyces stipitis JCM 10742T | Type strain | Japan Collection of Microorganisms, Japan |
Meyerozyma guilliermondii JCM 1539T | Type strain | Japan Collection of Microorganisms, Japan |
Brettanomyces bruxellensis CBS 2499 | Westerdijk Fungal Biodiversity Institute, The Netherlands | |
Schwanniomyces pseudopolymorphus JCM3652T | Type strain | Japan Collection of Microorganisms, Japan |
Schwanniomyces polymorphus var. polymorphus JCM3647T | Type strain | Japan Collection of Microorganisms, Japan |
Schwanniomyces polymorphus var. africanus JCM7443T | Type strain | Japan Collection of Microorganisms, Japan |
Plasmids | Feature Important for This Study | Reference |
pGP564 | contains 2µ replication origin | [12] |
pRS40B * | source of the pRS40 backbone | [13] |
pRS50 series (pRS50oK, pRS50oH, pRS50oN, pRS50oB) | Integrative plasmids with codon-optimized selectable markers (KanR, HygR, NarR, PhlR) | This study |
pRS52 series (pRS52oK, pRS52oH, pRS52oN, pRS52oB) | Contains 2μ replication origin from S. cerevisiae and codon-optimized selectable markers (KanR, HygR, NarR, PhlR) | This study |
pRS53 series (pRS53oK, pRS53oH, pRS53oN, pRS53oB) | Contains panARS replication origin from K. lactis and codon-optimized selectable markers (KanR, HygR, NarR, PhlR) | This study |
pRS54 series (pRS54oK, pRS54oH, pRS54oN, pRS54oB) | Contains SsARS replication origin from S. stipitis and codon-optimized selectable markers (KanR, HygR, NarR, PhlR) | This study |
pRS55 series (pRS55oK, pRS55oH, pRS55oN, pRS55oB) | Contains MgALS123 replication origin from M. guilliermondii and codon-optimized selectable markers (KanR, HygR, NarR, PhlR) | This study |
pRS56 series (pRS56oK, pRS56oH, pRS56oN, pRS56oB) | Contains BbCEN2 replication origin from B. bruxellensis and codon-optimized selectable markers (KanR, HygR, NarR, PhlR) | This study |
Oligonucleotides | Sequence | Reference |
panARS-f | gtgaggtaccgaaggaatttgctgttatggag | This study |
panARS-r | gtgaggtaccactgacactgttgactctg | This study |
SsARS2-f | gatatccagaataattgatggtccgc | This study |
SsARS2-r | gatatctggattgttgtgctctcg | This study |
MgARS-f | gatatcagatgacaagcccaaacac | This study |
MgARS-r | gatatccatatgtccttgccagttgaacca | This study |
DbCEN2-f | gatatcctgaggttgctaagcccc | This study |
DbCEN2-r | gatatcgtgaatagtgaagccaactggt | This study |
AgTEF-f | aggcctcccgggacatggaggcccagaat | This study |
AgTEF-r | aggcctcccgggcagtatagcgaccagcattc | This study |
Transformation Efficiency (Transformants/μg) | ||||||
---|---|---|---|---|---|---|
pRS50oH/SacII (Linear) | pRS52oH (2μ Origin) | pRS53oH (panARS Origin) | pRS54oH (SsARS2 Origin) | pRS55oH (MgALS123 Origin) | pRS56oH (BbCEN2 Origin) | |
S. pseudopolymorphus | 8.1 × 103 | 424 | 21 | 4.7 × 105 | 32 | 1.1 × 103 |
S. polymorphus var. polymorphus | 952 | 360 | 117 | 3.7 × 105 | More than 2 × 104 | 714 |
S. polymorphus var. africanus | 476 | 88 | 149 | 2.7 × 105 | More than 2 × 104 | 0 |
Plasmid Stability | |||
---|---|---|---|
pRS50oH Integrated in the Genome | pRS54oH (SsARS2 Origin) | pRS55oH (MgALS123 Origin) | |
S. pseudopolymorphus | 100% | 5.9% | Not replicative |
S. polymorphus var. polymorphus | 100% | 28% | 1% |
S. polymorphus var. africanus | 100% | 100% | 0.2% |
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Matanović, A.; Arambašić, K.; Žunar, B.; Štafa, A.; Svetec Miklenić, M.; Šantek, B.; Svetec, I.-K. Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus. J. Fungi 2022, 8, 531. https://doi.org/10.3390/jof8050531
Matanović A, Arambašić K, Žunar B, Štafa A, Svetec Miklenić M, Šantek B, Svetec I-K. Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus. Journal of Fungi. 2022; 8(5):531. https://doi.org/10.3390/jof8050531
Chicago/Turabian StyleMatanović, Angela, Kristian Arambašić, Bojan Žunar, Anamarija Štafa, Marina Svetec Miklenić, Božidar Šantek, and Ivan-Krešimir Svetec. 2022. "Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus" Journal of Fungi 8, no. 5: 531. https://doi.org/10.3390/jof8050531