title: Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form.
abstract: Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5'-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 microgram pRPPyr4 DNA and 1 x 10(7) viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0-1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.
authors.affiliation: Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yamazaki
authors.forename: Y
authors.initials: Y
authors.lastname: Ohnishi
authors.forename: K
authors.initials: K
authors.lastname: Takeuchi
authors.forename: N
authors.initials: N
authors.lastname: Mori
authors.forename: N
authors.initials: N
authors.lastname: Shiraishi
authors.forename: Y
authors.initials: Y
authors.lastname: Sakata
authors.forename: H
authors.initials: H
authors.lastname: Suzuki
authors.forename: S
authors.initials: S
authors.lastname: Horinouchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D015825:Chromosomes, Fungal / Q000235:genetics; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018628:Gene Dosage; D005816:Genetic Complementation Test; D006031:Glycosylation; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D008892:Milk / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D020103:Rhizomucor / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic; D014498:Uracil / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014498:Uracil; D001216:Asparagine; C083733:ALG2 protein, S cerevisiae; D008364:Mannosyltransferases; D016282:Aspartic Acid Endopeptidases; C022517:rennin-like enzyme (Aspergillus ochraceus); D009964:Orotidine-5'-Phosphate Decarboxylase; D008358:Mannose