13 C 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.
13 C 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.
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在有氧分批培养物中以葡萄糖为基础生长的毕赤酵母的 13 C 代谢通量分析显示，TCA 循环的相对合成代谢使用较高，并且所提供的支链氨基酸前体的掺入有限。
DOI：
10.1111/febs.14180
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Szyperski,Thomas
中科院分区：
文献类型：
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作者：
Zhang,Meng;Yu,Xiao-Wei;Xu,Yan;Jouhten,Paula;Swapna,GurlaVT;Glaser,RalfW;Hunt,JohnF;Montelione,GaetanoT;Maaheimo,Hannu;Szyperski,Thomas
利用13 C核磁共振（NMR）技术研究了克拉布特里阴性酵母毕赤酵母（Pichia pastoris）的碳代谢，以描述其指数生长过程中的调控，并研究了支链氨基酸生物合成的两种前体α-酮异戊酸和α-酮丁酸的输入。细胞在有氧分批培养物中生长，所述培养物含有（a）仅葡萄糖，（B）葡萄糖沿着前体，或（c）葡萄糖和瓦尔。这项研究提供了以下新的见解。首先，中心代谢的13 C通量比分析揭示了Crabtree阴性酵母的三羧酸循环的出乎意料的高回补供应，并表明相当大一部分葡萄糖催化剂通过戊糖磷酸途径进行。与先前对克拉布特里阴性的树干毕赤酵母和克拉布特里阳性的酿酒酵母分批培养的通量比分析的比较表明，在P. pastorisis intermediate在P. stipitisandS.啤酒。第二，培养基中过量的α-酮异戊酸不能转运到细胞质中，表明P.巴斯德缺乏合适的转运蛋白。相反，过量的瓦尔被有效地吸收，并在很大程度上满足了蛋白质合成对瓦尔和Leu的需求。第三，过量的α-酮丁酸被转运到线粒体中用于Ile生物合成。然而，输入不能有效地抑制丙酮酸合成α-酮丁酸，表明P. pastorishas没有进化优化充分利用这一碳源。这些发现对于制备均匀的2 H，13 C，15 N标记的蛋白质具有直接的意义，这些蛋白质含有质子化的Ile，瓦尔和Leu甲基基团。pastorisfor NMR-based structural biology.酶乙酰羟酸异构还原酶（EC 1.1.1.86），支链氨基酸转氨酶（BCAT，EC 2.6.1.42），（EC www.example.com），苹果酸酶（EC 1.1.1.39/1.1.1.40），磷酸烯醇丙酮酸羧激酶（EC 4.1.1.49），丙酮酸羧化酶（EC 6.4.1.1），丙酮酸激酶（EC 2.7.1.40），l-丝氨酸羟甲基转移酶（EC 2.1.2.1），苏氨酸醛缩酶（EC 4.1.2.5），苏氨酸脱氢酶（EC 4.3.1.19）;转酮醇酶（EC2.2.1.2），转醛醇酶（EC 2.2.1.1）。4.2.1.2
Carbon metabolism of Crabtree‐negative yeastPichia pastoriswas profiled using13C 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,13C 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‐negativePichia stipitisand Crabtree‐positiveSaccharomyces cerevisiaeindicate that the overall regulation of central carbon metabolism inP. pastorisis intermediate in betweenP. stipitisandS. cerevisiae. Second, excess α‐ketoisovalerate in the medium is not transported into the cytoplasm indicating thatP. pastorislacks 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 thatP. pastorishas not been optimized evolutionarily to take full advantage of this carbon source. These findings have direct implications for preparing uniformly2H,13C,15N‐labeled proteins containing protonated Ile, Val, and Leu methyl groups inP. pastorisfor NMR‐based structural biology.EnzymesAcetohydroxy 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).