THREONINE ALDOLASE OVEREXPRESSION PLUS THREONINE SUPPLEMENTATION ENHANCED RIBOFLAVIN PRODUCTION IN ASHBYA-GOSSYPII

Riboflavin production in the filamentous fungus Ashbya gossypii is lim ited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overe xpression of the glycine biosynthetic enzyme threonine aldolase. The G LY1 gene encoding the threonine aldolase of A. gossypii was isolated b y heterologous complementation of the glycine-auxotrophic Saccharomyce s cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg(-1) was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not a ffected. This indicated a minor role of the enzyme in glycine biosynth esis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold incr ease of threonine aldolase specific activity in crude extracts along w ith a 9-fold increase of riboflavin production when the medium was sup plemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an a lmost quantitative uptake of threonine and its intracellular conversio n into glycine. This became evident by a subsequent partial efflux of the glycine formed.