Highly-efficient electrotransformation of the yeast Hansenula polymorpha

Faber, Klaas Nico; Haima, Peter; Harder, W.; Veenhuis, Marten; Ab, Geert

doi:10.1007/bf00351482

Cited by 258 publications

(202 citation statements)

References 24 publications

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“…S8) and made by Integrated DNA Technologies. The construct was inserted into a panARS vector (63) containing a KanMX marker by restriction digestion and ligation and was transformed into H. polymorpha by electroporation as previously described (64). Log-phase cells were crosslinked using formaldehyde (1% final concentration) for 20 min followed by the addition of glycine to stop the reaction.…”

Section: Methodsmentioning

confidence: 99%

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locusSaccharomyces cerevisiaesystem

Hanson

Byrne

Wolfe

2014

Proc. Natl. Acad. Sci. U.S.A.

150

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Saccharomyces cerevisiae has a complex system for switching the mating type of haploid cells, requiring the genome to have three mating-type (MAT)-like loci and a mechanism for silencing two of them. How this system originated is unknown, because the threelocus system is present throughout the family Saccharomycetaceae, whereas species in the sister Candida clade have only one locus and do not switch. Here we show that yeasts in a third clade, the methylotrophs, have a simpler two-locus switching system based on reversible inversion of a section of chromosome with MATa genes at one end and MATalpha genes at the other end. In Hansenula polymorpha the 19-kb invertible region lies beside a centromere so that, depending on the orientation, either MATa or MATalpha is silenced by centromeric chromatin. In Pichia pastoris, the orientation of a 138-kb invertible region puts either MATa or MATalpha beside a telomere and represses transcription of MATa2 or MATalpha2. Both species are homothallic, and inversion of their MAT regions can be induced by crossing two strains of the same mating type. The three-locus system of S. cerevisiae, which uses a nonconservative mechanism to replace DNA at MAT, likely evolved from a conservative two-locus system that swapped genes between expression and nonexpression sites by inversion. The increasing complexity of the switching apparatus, with three loci, donor bias, and cell lineage tracking, can be explained by continuous selection to increase sporulation ability in young colonies. Our results provide an evolutionary context for the diversity of switching and silencing mechanisms.Hansenula polymorpha | Pichia pastoris | yeast genetics | comparative genomics M ating-type switching in yeasts is a highly regulated process that converts a haploid cell of one mating type into a haploid of the opposite type (1-5). Switching involves the complete deactivation of one set of regulatory genes and activation of an alternative set, but, unlike most regulatory changes, the switch is achieved by physically replacing the DNA at an expression site that is shared by both types of cell. In Saccharomyces cerevisiae the switching system uses a menagerie of molecular components (3) including three mating-type (MAT)-like loci (the expressed MAT locus and the silent loci HML and HMR); an endonuclease (HO) that creates a double-strand break at the MAT locus, which then is repaired using HMLalpha or HMRa as a donor; a mechanism (Sir1 and Sir2/3/4 proteins) for repressing transcription and HO cleavage at the silent loci; two triplicated sequences (the Z and X regions) that guide repair of the dsDNA break; a donorbias mechanism (the recombination enhancer, RE) to ensure that switching happens in the correct direction; and a cell lineagetracking mechanism (Ash1 mRNA localization) to ensure that switching occurs only in particular cells. Most of these components have no function other than facilitating switching.

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Section: Methodsmentioning

confidence: 99%

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locusSaccharomyces cerevisiaesystem

Hanson

Byrne

Wolfe

2014

Proc. Natl. Acad. Sci. U.S.A.

150

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“…Transformation of H. polymorpha was performed by electroporation (Faber et al, 1994b). Restriction enzymes and biochemicals were obtained from Roche (Almere, The Netherlands) and used as detailed by the manufacturer.…”

Section: Molecular Techniquesmentioning

confidence: 99%

Routing ofHansenula polymorphaAlcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery

Gunkel

Dijk

Veenhuis

et al. 2004

MBoC

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Import of Hansenula polymorpha alcohol oxidase (AO) into peroxisomes is dependent on the PTS1 receptor, HpPex5p. The PTS1 of AO (-LARF) is sufficient to direct reporter proteins to peroxisomes. To study AO sorting in more detail, strains producing mutant AO proteins were constructed. AO containing a mutation in the FAD binding fold was mislocalized to the cytosol. This indicates that the PTS1 of AO is not sufficient for import of AO. AO protein in which the PTS1 was destroyed (؊LARA) was normally sorted to peroxisomes. Moreover, C-terminal deletions of up to 16 amino acids did not significantly affect AO import, indicating that the PTS1 was not necessary for targeting. Consistent with these observations we found that AO import occurred independent from the C-terminal TPR-domain of HpPex5p, known to bind PTS1 peptides. Synthesis of the N-terminal domain (amino acids 1-272) of HpPex5p in pex5 cells restored AO import, whereas other PTS1 proteins were mislocalized to the cytosol. These data indicate that AO is imported via a novel HpPex5p-dependent protein translocation pathway, which does not require the PTS1 of AO and the C-terminal TPR domains of HpPex5p, but involves FAD binding and the N-terminus of HpPex5p.

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“…Efficient and reliable transformation procedures for H. polymorpha by using electroporation have been developed by Faber et al [6] (transformation frequency of 1.7 ϫ 10 6 / g DNA). Optimal results are obtained by using batch cells from the midexponential growth phase that are pretreated with the reducing agent DTT to weaken the cell wall.…”

Section: Transformation Proceduresmentioning

confidence: 99%