New selectable host-marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, Korea.
Yeast (Impact Factor: 1.63). 09/2009; 26(9):507-21. DOI: 10.1002/yea.1701
Source: PubMed

ABSTRACT Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively.

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    • "The presence of an active OAS pathway in H. polymorpha was further supported by analysis of the growth phenotypes of the HpSAT1 disruptant (Hpsat1Δ), which were clearly distinctive from those of the HpMET2 disruptant (Hpmet2Δ) with deletion of the HpMET2 gene encoding homoserine O-acetyltransferase [24]. While growth of Hpsat1Δ on B-medium can be supported by supplementation with cysteine or methionine, growth of Hpmet2Δ is strictly dependent on the addition of methionine (Fig. 4C). "
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    ABSTRACT: In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species.
    PLoS ONE 06/2014; 9(6):e100725. DOI:10.1371/journal.pone.0100725 · 3.23 Impact Factor
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    • "Although the rate of tryptophan auxotrophs obtained among FAA-resistant clones remains low in these experiments (10–30 %), it has previously been observed in several other species (Cheon et al. 2009). In fungal species, the percentages of tryptophan auxotroph among FAA-resistant clones are highly variable ranging from 5–8 % in Hansenula polymorpha (Cheon et al. 2009) to 100 % in S. cerevisiae (Toyn et al. 2000). Therefore, it is possible that in C. guilliermondii , FAA resistance could be mediated predominantly by the activation of a compensatory biosynthetic pathway(s). "
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    ABSTRACT: Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3'UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast "TRP blaster" disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created "TRP5 blaster" disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.
    Current Genetics 05/2012; 58(4):245-54. DOI:10.1007/s00294-012-0377-3 · 2.68 Impact Factor
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    • "This study pDTMOX-msdS PMOX-MsdS-HA-HDEL, HpTRP1-HARS36 Cheon et al. (2009) pDTG-msdS PGAP-MsdS-HA-HDEL, HpTRP1-HARS36 This study pDLGAP-ScCPY PGAP-ScCPY, HpLEU2-HARS36 This study a MGAT1 contains the DNA fragment of 407 amino acids from the C-terminus of human GnTI (GenBank number NM_002406); ScMNN9s and ScMNN9m contain the DNA fragment encoding 32 amino acids and 40 amino acids from its N-terminus, respectively; HpOCH1s and HpOCR1m contain the DNA fragment encoding 52 amino acids and 46 amino acids from its N-terminus, respectively. taining the methanol-inducible MOX promoter and the Zeocin resistance marker (Fig. 1B) was created by replacing the BglII-XbaI GAP promoter fragment in pGAPZαA (Invitrogen) with the 1.5 kb MOX promoter fragment, which was PCR-amplified with a primer set HP01/HP02 from H. polymorpha genomic DNA. "
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    ABSTRACT: As a step forward to achieve the generation of human complex-type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc(1)Man(5)GlcNAc(2) and GlcNAc(1)Man(3)GlcNAc(2), respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.
    The Journal of Microbiology 04/2012; 50(2):341-8. DOI:10.1007/s12275-012-2097-2 · 1.44 Impact Factor
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