TAILIEUCHUNG - Báo cáo khoa học: Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris

Amino acid biosynthesis and central carbon metabolism of Pichia pastoriswere studied using biosynthetically directed fractional 13 C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates ( h )1 , h )1 ) with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations, cells were also grown at h )1 on glucose. Our results show that, firstly, amino acids are synthesized as inSaccharomyces cerevisiae. . | Eur. J. Biochem. 271 2462-2470 2004 FEBS 2004 doi Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris Aina Sola1 2 Hannu Maaheimo2 3. Katri Ylonen3. Pau Ferrer1 and Thomas Szvnerski2 1 Department of Chemical Engineering Escola Tecnica Superior d Enginyeria Universitat Autonoma de Barcelona Bellaterra Spain 2Department of Chemistry University at Buffalo The State University of New York at Buffalo NY USA 3NMR-laboratory and Structural Biology and Biophysics Program VTT Biotechnology Helsinki Finland Amino acid biosynthesis and central carbon metabolism of Pichia pastoris were studied using biosynthetically directed fractional 13C labeling. Cells were grown aerobically in a chemostat culture fed at two dilution rates h-1 h-1 with glycerol as the sole carbon source. For investigation of amino acid biosynthesis and comparison with glycerol cultivations cells were also grown at h-1 on glucose. Our results show that firstly amino acids are synthesized as in Saccharomyces cerevisiae. Secondly biosynthesis of mitochondrial pyruvate via the malic enzyme is not registered for any of the three cultivations. Thirdly transfer of oxaloacetate across the mitochondrial membrane appears bidirectional with a smaller fraction of cytosolic oxaloacetate stemming from the mitochondrial pool at the higher dilution rate of h-1 for glucose or glycerol cul tivation when compared to the glycerol cultivation at h-1. Fourthly the fraction of anaplerotic synthesis of oxaloacetate increases from 33 to 48 when increasing the dilution rate for glycerol supply while 38 is detected when glucose is fed. Finally the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S. cerevisiae grown on glucose in a chemostat culture at a dilution rate of h-1. This provided a first indication that regulation of central carbon metabolism in P. .

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