Departament d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain.
N Biotechnol. 2014 Jan 25;31(1):120-32. doi: 10.1016/j.nbt.2013.06.007. Epub 2013 Jul 8.
The yeast Pichia pastoris has emerged as one of the most promising yeast cell factories for the production of heterologous proteins. The readily available genetic tools and the ease of high-cell density cultivations using methanol or glycerol/methanol mixtures are among the key factors for this development. Previous studies have shown that the use of mixed feeds of glycerol and methanol seem to alleviate the metabolic burden derived from protein production, allowing for higher specific and volumetric process productivities. However, initial studies of glycerol/methanol co-metabolism in P. pastoris by classical metabolic flux analyses using (13)C-derived Metabolic Flux Ratio (METAFoR) constraints were hampered by the reduced labelling information obtained when using C3:C1 substrate mixtures in relation to the conventional C6 substrate, that is, glucose. In this study, carbon flux distributions through the central metabolic pathways in glycerol/methanol co-assimilation conditions have been further characterised using biosynthetically directed fractional (13)C labelling. In particular, metabolic flux distributions were obtained under 3 different glycerol/methanol ratios and growth rates by iterative fitting of NMR-derived (13)C-labelling data from proteinogenic amino acids using the software tool (13)CFlux2. Specifically, cells were grown aerobically in chemostat cultures fed with 80:20, 60:40 and 40:60 (w:w) glycerol/methanol mixtures at two dilutions rates (0.05 hour(-1) and 0.16 hour(-1)), allowing to obtain additional data (biomass composition and extracellular fluxes) to complement pre-existing datasets. The performed (13)C-MFA reveals a significant redistribution of carbon fluxes in the central carbon metabolism as a result of the shift in the dilution rate, while the ratio of carbon sources has a lower impact on carbon flux distribution in cells growing at the same dilution rate. At low growth rate, the percentage of methanol directly dissimilated to CO2 ranges between 50% and 70%. At high growth rate the methanol is completely dissimilated to CO2 by the direct pathway, in the two conditions of highest methanol content.
毕赤酵母已成为生产异源蛋白最有前途的酵母细胞工厂之一。其易于获得的遗传工具以及使用甲醇或甘油/甲醇混合物进行高密度细胞培养的便利性是其发展的关键因素之一。先前的研究表明，使用甘油和甲醇的混合饲料似乎可以减轻蛋白生产带来的代谢负担，从而提高比和体积产率。然而，使用基于（13）C 的代谢通量比（METAFoR）约束的经典代谢通量分析对毕赤酵母中甘油/甲醇共代谢的初步研究受到了限制，因为与传统的 C6 底物（即葡萄糖）相比，使用 C3：C1 底物混合物时获得的标记信息减少了。在这项研究中，通过生物合成定向的分数（13）C 标记进一步研究了甘油/甲醇共同化条件下中央代谢途径的碳通量分布。特别是，通过使用软件工具（13）CFlux2 对源自 NMR 的（13）C 标记数据进行迭代拟合，获得了在 3 种不同的甘油/甲醇比和生长速率下的代谢通量分布。具体来说，细胞在恒化器培养物中进行好氧生长，以 80:20、60:40 和 40:60（w:w）的甘油/甲醇混合物进料，在两个稀释率（0.05 小时（-1）和 0.16 小时（-1））下进行培养，以获得补充现有数据集的额外数据（生物量组成和细胞外通量）。进行的（13）C-MFA 表明，由于稀释率的变化，中央碳代谢中的碳通量发生了显著的重新分配，而碳源的比例对在相同稀释率下生长的细胞中碳通量分布的影响较小。在低生长速率下，甲醇直接分解为 CO2 的百分比在 50%至 70%之间。在高生长速率下，甲醇通过直接途径完全分解为 CO2，在甲醇含量最高的两种条件下都是如此。
