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


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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Available from: Seon Ah Cheon, Sep 30, 2015
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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.
    Full-text · Article · Jun 2014 · PLoS ONE
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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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    Full-text · Article · May 2012 · Current Genetics
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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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    Full-text · Article · Apr 2012 · The Journal of Microbiology
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