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Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

ABSTRACT: Riboflavin production in the filamentous fungus Ashbya gossypii is 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. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces 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 affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter 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.

SUBMITTER: Monschau N

PROVIDER: S-EPMC106640 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

Monschau N N Sahm H H Stahmann K K

Applied and environmental microbiology 19981101 11

Riboflavin production in the filamentous fungus Ashbya gossypii is 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. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced am ...[more]

PMID: 9797278

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Project description:BackgroundThe fungus Ashbya gossypii is an important industrial producer of the vitamin riboflavin. Using this microbe, riboflavin is manufactured in a two-stage process based on a rich medium with vegetable oil, yeast extract and different precursors: an initial growth and a subsequent riboflavin production phase. So far, our knowledge on the intracellular metabolic fluxes of the fungus in this complex process is limited, but appears highly relevant to better understand and rationally engineer the underlying metabolism. To quantify intracellular fluxes of growing and riboflavin producing A. gossypii, studies with different 13C tracers were embedded into a framework of experimental design, isotopic labeling analysis by MS and NMR techniques, and model-based data processing. The studies included the use 13C of yeast extract, a key component used in the process.ResultsDuring growth, the TCA cycle was found highly active, whereas the cells exhibited a low flux through gluconeogenesis as well as pentose phosphate pathway. Yeast extract was the main carbon donor for anabolism, while vegetable oil selectively contributed to the proteinogenic amino acids glutamate, aspartate, and alanine. During the subsequent riboflavin biosynthetic phase, the carbon flux through the TCA cycle remained high. Regarding riboflavin formation, most of the vitamin's carbon originated from rapeseed oil (81 ± 1%), however extracellular glycine and yeast extract also contributed with 9 ± 0% and 8 ± 0%, respectively. In addition, advanced yeast extract-based building blocks such as guanine and GTP were directly incorporated into the vitamin.ConclusionIntracellular carbon fluxes for growth and riboflavin production on vegetable oil provide the first flux insight into a fungus on complex industrial medium. The knowledge gained therefrom is valuable for further strain and process improvement. Yeast extract, while being the main carbon source during growth, contributes valuable building blocks to the synthesis of vitamin B2. This highlights the importance of careful selection of the right yeast extract for a process based on its unique composition.

Dataset Information

Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

Publications

Threonine aldolase overexpression plus threonine supplementation enhanced riboflavin production in Ashbya gossypii.

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