13C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched‐chain amino acids. (7th August 2017) - Zhang, Meng
Abstract : Carbon metabolism of Crabtree‐negative yeast Pichia pastoris was profiled using 13 C nuclear magnetic resonance (NMR) to delineate regulation during exponential growth and to study the import of two precursors for branched‐chain amino acid biosynthesis, α‐ketoisovalerate and α‐ketobutyrate. Cells were grown in aerobic batch cultures containing (a) only glucose, (b) glucose along with the precursors, or (c) glucose and Val. The study provided the following new insights. First, 13 C flux ratio analyses of central metabolism reveal an unexpectedly high anaplerotic supply of the tricarboxylic acid cycle for a Crabtree‐negative yeast, and show that a substantial fraction of glucose catabolism proceeds through the pentose phosphate pathway. A comparison with previous flux ratio analyses for batch cultures of Crabtree‐negative Pichia stipitis and Crabtree‐positive Saccharomyces cerevisiae indicate that the overall regulation of central carbon metabolism in P. pastoris is intermediate in between P. stipitis and S. cerevisiae . Second, excess α‐ketoisovalerate in the medium is not transported into the cytoplasm indicating that P. pastoris lacks a suitable transporter. In contrast, excess Val is efficiently taken up and largely fulfills demands for both Val and Leu for protein synthesis. Third, excess α‐ketobutyrate is transported into the mitochondria for Ile biosynthesis. However, the import does not efficiently inhibit the synthesis of α‐ketobutyrate from pyruvate indicating that P. pastoris has not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly 2 H, 13 C, 15 N‐labeled proteins containing protonated Ile, Val, and Leu methyl groups in P. pastoris for NMR‐based structural biology. Enzymes: Acetohydroxy acid isomeroreductase (EC 1.1.1.86 ), branched‐chain amino acid aminotransferase (BCAT, EC 2.6.1.42 ), fumarase (EC 4.2.1.2 ), malic enzyme (EC 1.1.1.39 /1.1.1.40 ), phosphoenolpyruvate carboxykinase (EC 4.1.1.49 ), pyruvate carboxylase (EC 6.4.1.1 ), pyruvate kinase (EC 2.7.1.40 ), l‐ serine hydroxymethyltransferase (EC 2.1.2.1 ), threonine aldolase (EC 4.1.2.5 ), threonine dehydratase (EC 4.3.1.19 ); transketolase (EC 2.2.1.1 ), transaldolase (EC 2.2.1.2 ). Abstract : 13 C NMR‐based metabolic profiling of the yeast Pichia pastoris in aerobic batch cultures containing glucose and precursors of branched‐chain amino acids reveals distinct features of the overall regulation of central carbon metabolism, which is intermediate between Pichia stipitis and Saccharomyces cerevisiae, as well as insights into limitations of α‐ketoisovalerate and α‐ketobutyrate transport from the medium into cytosol and mitochondria.