Improved Anaerobic Use of Arginine by Saccharomyces cerevisiae

Abstract

Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding ⌬ 1 -pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.

Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae

“…In agreement with that notion, we recently reported that a substantial fraction of arginine, once transported into the cell, had accumulated inside the vacuole before being metabolized during the last stages of the growth phase (33). When arginine started to be substantially consumed (N ¾ ), the isotopic enrichment of glutamate, an intermediate of the arginine assimilation pathway (17,35), increased over that at the first stage of growth, while the level of labeling in other amino acids remained very low (Fig. 2D); at the end of the growth phase (N T ), nitrogen from arginine was equally redistributed between proteinogenic amino acids.…”

Engineering of Yeast Put4 Permease and Its Application to Lager Yeast for Efficient Proline Assimilation

“…39) Some genetic approaches have been considered with wine yeast in order to improve the assimilation of arginine and proline, which is repressed by nitrogen catabolite repression triggered by ammonia in the grape must. 40,41) It is our hope that the establishment of means for efficient utilization of less assimilable amino acids will facilitate the production of a variety of novel alcoholic beverages. …”

Proline transport and stress tolerance of ammonia-insensitive mutants of the PUT4-encoded proline-specific permease in yeast

“…Under anaerobic conditions, yeast is able to assimilate only three of the four nitrogen atoms of arginine with the fourth, as proline, being unassimilated. Martin et al (2003) were however able to modify the anaerobic degradation of arginine, to allow for the assimilation of all four nitrogen atoms. That is, the NADPH-dependent P5C reductase (PRO3) and the URE2 negative regulator were deleted, while a PUT2 (encoding NAD(P) linked Δ 1 -pyrroline-5-carboxylate dehydrogenase) devoid of a mitochondrial targeting sequence was strongly expressed (see Fig.1).…”