Use of the pyrG gene as a food-grade selection marker in Monascus.
Ma-pyrG was cloned from Monascus aurantiacus AS3.4384 using degenerate PCR with primers designed with an algorithm called CODEHOP, and its complete sequence was obtained by a PCR-based strategy for screening a Monascus fosmid library. Ma-pyrG encodes orotidine-5'-phosphate decarboxylase (OMPdecase), a 283-aminoacid protein with 81% sequence identity to that from Aspergillus flavus NRRL 3357. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +220. Plasmid pGFP-pyrG, bearing the green fluorescent protein gene (GFP) as a model gene and Ma-pyrG as a selection marker, were constructed. pGFP-pyrG were successfully transformed into UM28 by using the PEG method.
Wang
Bo-hua
BH
State Key Laboratory of Food Science and Technology, Nanchang University, Nanjing East Road 235, Nanchang, Jiangxi 330047, China.
Xu
Yang
Y
Li
Yan-ping
YP
D001231:Aspergillus flavus / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008969:Molecular Sequence Data; D032741:Monascus / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification / Q000528:radiation effects; D016296:Mutagenesis; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000633:toxicity; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D009964:Orotidine-5'-Phosphate Decarboxylase