title: A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.
abstract: The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.
authors.affiliation: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, South Korea.
authors.forename: Hye Yun
authors.initials: HY
authors.lastname: Moon
authors.affiliation: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, South Korea.
authors.forename: Dong Wook
authors.initials: DW
authors.lastname: Lee
authors.affiliation: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, South Korea.
authors.forename: Gyu Hun
authors.initials: GH
authors.lastname: Sim
authors.affiliation: College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea.
authors.forename: Hong-Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Pathology Division, National Institute of Fisheries Science (NIFS), Busan 46083, South Korea.
authors.forename: Jee Youn
authors.initials: JY
authors.lastname: Hwang
authors.affiliation: Pathology Division, National Institute of Fisheries Science (NIFS), Busan 46083, South Korea.
authors.forename: Mun-Gyeong
authors.initials: MG
authors.lastname: Kwon
authors.affiliation: Green Cross Veterinary Products Co. LTD., Yongin, Kyunggi-Do, South Korea.
authors.forename: Bo-Kyu
authors.initials: BK
authors.lastname: Kang
authors.affiliation: Green Cross Veterinary Products Co. LTD., Yongin, Kyunggi-Do, South Korea.
authors.forename: Jong Man
authors.initials: JM
authors.lastname: Kim
authors.affiliation: Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, South Korea; Bio-Integration Research Center for Nutra-Pharmaceutical Epigenetics, Chung-Ang University, Seoul 156-756, South Korea. Electronic address: hyunkang@cau.ac.kr.
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kang
mesh_terms: D000818:Animals; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005260:Female; D005818:Genetic Engineering / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D021903:DNA, Ribosomal Spacer; D011994:Recombinant Proteins
keywords: Defective auxotrophic markers; Multiple integration; Recombinant yeast; Ribosomal DNA non-transcribed spacer