A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1.
To facilitate the selection of multiple gene integrants in Hansenula polymorpha, a rapid and copy-number-controlled selection system was developed using a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the unmodified APH gene as a dominant selectable marker resulted in the extremely slow growth of transformants and the frequent selection of spontaneous resistance. For the proper performance of the APH gene, a set of deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene. The fusion construct with the 578-bp GAPDH promoter conferred G418 resistance sufficient to allow rapid growth of transformants, and thus facilitated the selection of transformants with up to 15 tandem copies of the vector. To increase further the integration copy number within the gene-dose-dependent range, the GAPDH promoter was serially deleted down to the -61 nucleotide. With this weak expression cassette, the integration copy number could easily be controlled between 1 and 50. Tandemly integrated copies of plasmids near the end of the chromosome were mitotically stable over 150 generations. The dosage-dependent selection system of this study would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.
Sohn
J H
JH
Biotechnology Research Division, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea.
Choi
E S
ES
Kang
H A
HA
Rhee
J S
JS
Agaphonov
M O
MO
Ter-Avanesyan
M D
MD
Rhee
S K
SK
D001426:Bacterial Proteins; D015152:Blotting, Northern; D015139:Blotting, Southern; D004261:DNA Replication; D004251:DNA Transposable Elements; D017353:Gene Deletion; D018628:Gene Dosage; D005800:Genes, Fungal; D005819:Genetic Markers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D005819:Genetic Markers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C106858:spectinomycin phosphotransferase