A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1

Abstract

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.

Glucose‐induced production of recombinant proteins in Hansenulapolymorpha mutants deficient in catabolite repression

“…As an example, the vector used for isolation of HBsAg producers, pKO6, supports multiple tandem integration into telomeric chromosome region (Sohn et al, 1999). Cassette copy number, therefore, can be established by serial dilutions of genomic DNA and comparing to untransformed strain's single-copy signal of the H. polymorpha-LEU2 gene.…”

“…The HindIII flanked PCR fragment was further subcloned onto plasmid pET1 (Tan et al, 1995) to place the HBsAg gene between P MOX and MOX terminator, producing plasmid pOS56. BamHI/SspI fragment from plasmid pOS56, harboring entire HBsAg expression cassette, was ligated into BamHI/ SacII digested pGLG61 multiple integration vector (Sohn et al, 1999; kindly provided by Dr. H. A. Kang) resulting in plasmid pKO6 used in this study. pKO6 harbors the HpLEU2 gene and bacterial APH (geneticine (G418)-resistance) gene placed under short version of constitutive GAP (glyceraldehyde-3-phosphate dehydrogenase) gene promoter for positive selection in H. polymorpha.…”

“…This HindIII flanked fragment was further subcloned onto plasmid pHIPX2 (Faber et al, 1994b), to place the MFa-MPI fusion gene between P MOX and terminator of the HpAMO gene, producing plasmid pHIns. BamHI/BglII fragment from the latter plasmid, harboring the entire MPI expression cassette, was ligated into BamHI digested vector pKO12, derivative of pGLG61 multiple integration vector (Sohn et al, 1999), resulting in plasmid pKO13. pKO12 carries an alternative selection marker, the H. polymorpha formaldehyde dehydrogenase (FLD1) gene (Baerends et al, 2002), placed under the shortened GAP promoter instead of the APH gene.…”

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

“…DNA sequencing was carried out with an automatic DNA sequencer (ABI model 373A; Applied Biosystems). The plasmid pGA-GOD-CwpF (15) contained the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene promoter (27), HARS36 for autonomous replication (28), the LEU2 gene of H. polymorpha (2), and CwpF as a surface display anchor from H. polymorpha (15). This plasmid was used as a backbone for construction of a Candida antarctica lipase B (CALB) expression vector.…”

“…The combination of the ARS domain and the telomeric repeats of HARS36 greatly increases the potential of HARS36 for multiple gene integration into the chromosome. The expression level of foreign proteins, however, is diverse due to a difference in the integrated gene copy number (3,27). Thus, the integrants are not adequate for activity-based selection of a mutant library.…”