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Title 

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

 

메탄올자화효모 한세눌라 폴리모파의 다중유전자 조작을 위한 신규 선별 숙주-표지 유전자 시스템

Authors 

S A CheonJ ChooV M UbiyvovkJ N ParkMoo Woong KimDoo-Byoung OhOh Suk KwonA A SibirnyJ Y KimH A Kang

Publisher 

Wiley-Blackwell

Issue Date 

2009

Citation 

Yeast, vol. 26, no. 9, pp. 507-521

Keywords 

ADE2Auxotrophic markerExpression vectorHansenula polymorphaMET2TRP1

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.

ISSN 

0749-503X

Link 

http://dx.doi.org/10.1002/yea.1701

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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