Expression of alanine:glyoxylate aminotransferase gene from Saccharomyces cerevisiae in Ashbya gossypii.
Two plasmids containing an autonomously replicating sequence from Saccharomyces cerevisiae were constructed. Using these vectors, the AGX1 gene encoding alanine:glyoxylate aminotransferase (AGT) from S. cerevisiae, which converts glyoxylate into glycine but is not present in Ashbya gossypii, was expressed in A. gossypii. Geneticin-resistant transformants with the plasmid having the kanamycin resistance gene under the control of the translation elongation factor 1 alpha (TEF) promoter and terminator from A. gossypii were obtained with a transformation efficiency of approximately 10-20 transformants per microgram of plasmid DNA. The specific AGT activities of A. gossypii pYPKTPAT carrying the AGX1 gene in glucose- and rapeseed-oil-containing media were 40 and 160 mU mg-1 of wet mycelial weight, respectively. The riboflavin concentrations of A. gossypii pYPKTPAT carrying AGX1 gene in glucose- and rapeseed-oil-containing media were 20 and 150 mg l-1, respectively. In the presence of 50 mM glyoxylate, the riboflavin concentration and the specific riboflavin concentration of A. gossypii pYPKTPAT were 2- and 1.3-fold those of A. gossypii pYPKT without the AGX1 gene.
Kato
Tatsuya
T
Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka, 422-8529, Japan.
Park
Enoch Y
EY
D000410:Alanine Transaminase / Q000096:biosynthesis* / Q000235:genetics; D001203:Ascomycota / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D005229:Fatty Acids, Monounsaturated; D005800:Genes, Fungal; D005818:Genetic Engineering; D005822:Genetic Vectors; D005947:Glucose; D010938:Plant Oils; D010957:Plasmids; D000074262:Rapeseed Oil; D011994:Recombinant Proteins / Q000096:biosynthesis; D012256:Riboflavin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D000637:Transaminases / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; D005229:Fatty Acids, Monounsaturated; D010938:Plant Oils; D000074262:Rapeseed Oil; D011994:Recombinant Proteins; D000637:Transaminases; C023067:glyoxylate aminotransferase; D000410:Alanine Transaminase; D005947:Glucose; D012256:Riboflavin