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

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. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1 ), FJ226453 (HpMET2 ) and FJ493241 (HpADE2 ), respectively.

Evaluating the influence of selection markers on obtaining selected pools and stable cell lines in human cells

“…No prior study has tested the efficacy of all of these selection agents in the same context and cell lines for a mammalian protein production host. Yet, analogous studies comparing performance differences have been carried out in other model eukaryotes including plants [28–30], yeasts [31, 32], and insects [33]. Selection marker choice is known to be a critical part of expression vector design [34, 35].…”

Novel Cysteine-Centered Sulfur Metabolic Pathway in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

“…The presence of an active OAS pathway in H. polymorpha was further supported by analysis of the growth phenotypes of the HpSAT1 disruptant ( Hpsat1 Δ), which were clearly distinctive from those of the HpMET2 disruptant ( Hpmet2 Δ) with deletion of the HpMET2 gene encoding homoserine O -acetyltransferase [24]. While growth of Hpsat1 Δ on B-medium can be supported by supplementation with cysteine or methionine, growth of Hpmet2 Δ is strictly dependent on the addition of methionine (Fig.…”

Deus ex Candida genetics: overcoming the hurdles for the development of a molecular toolbox in the CTG clade

“…This compound, toxic for prototrophic cells, permits in most cases an easy counterselection of ura3 or ura5 genotypes from a randomly mutagenized population. In the same way, recent advances have demonstrated the potential of 5-fluoroanthranilate for counter-selection of trp1, trp3, trp4 or trp5 genotypes in yeast (Toyn et al, 2000;Cheon et al, 2003Cheon et al, , 2009Lebel et al, 2006;Jones et al, 2008).…”