Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha

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Title
Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha
Author(s)
Kang Hyun Ah; Won Kyuong Hong; Jung Hoon Sohn; Eui Sung Choi; Sang Ki Rhee
Bibliographic Citation
Applied Microbiology and Biotechnology, vol. 55, no. 6, pp. 734-741
Publication Year
2001
Abstract
The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.
ISSN
0175-7598
Publisher
Springer
DOI
http://dx.doi.org/10.1007/s002530100605
Type
Article
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Division of Bio Technology Innovation > BioProcess Engineering Center > 1. Journal Articles
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