Threonine Aldolase Overexpression plus Threonine Supplementation Enhanced Riboflavin Production inAshbya gossypii

Abstract

ABSTRACTRiboflavin production in the filamentous fungusAshbya gossypiiis limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. TheGLY1gene encoding the threonine aldolase ofA. gossypiiwas isolated by heterologous complementation of the glycine-auxotrophicSaccharomyces cerevisiaestrain YM13 with a genomic library fromA. gossypii. The deduced amino acid sequence ofGLY1showed 88% similarity to threonine aldolase fromS. cerevisiae. In the presence of theGLY1gene, 25 mU of threonine aldolase specific activity mg−1was detectable in crude extracts ofS. cerevisiaeYM13. Disruption ofGLY1led to a complete loss of threonine aldolase activity inA. gossypiicrude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis ofA. gossypii. However, overexpression ofGLY1under the control of the constitutiveTEFpromoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.