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 polymo rpha, a rapid and copy-number-controlled selection system was developed usi ng a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the codified APH gene as a dominant selectable marker resulted in the extremel y slow growth of transformants and the frequent selection of spontaneous re sistance. For the proper performance of the APH gene, a set of deleted glyc eraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha wer e fused to the APH gene. The fusion construct with the 578-bp GAPDH promote r conferred G418 resistance sufficient to allow rapid growth of transforman ts, and thus facilitated the selection of transformants with up to 15 tande m copies of the vector. To increase further the integration copy number wit hin the gene-dose-dependent range, the GAPDH promoter was serially deleted down to the -61 nucleotide. With this weak expression cassette, the integra tion copy number could easily be controlled between 1 and 50. Tandemly inte grated copies of plasmids near the end of the chromosome were mitotically s table over 150 generations. The dosage-dependent selection system of this s tudy would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.