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首页> 外文期刊>New biotechnology >Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids
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Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids

机译：通过迭代拟合蛋白质来源的NMR衍生的13C标记数据，对在不同甘油/甲醇混合物上生长的重组毕赤酵母进行代谢通量分析

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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 13C-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 (0.05hour~(-1) and 0.16hour~(-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 CO_2 ranges between 50% and 70%. At high growth rate the methanol is completely dissimilated to CO_2 by the direct pathway, in the two conditions of highest methanol content.
机译：酵母巴斯德毕赤酵母已经成为生产异源蛋白质的最有希望的酵母细胞工厂之一。易于获得的遗传工具以及使用甲醇或甘油/甲醇混合物进行高细胞密度培养的简便性是这一发展的关键因素。以前的研究表明，甘油和甲醇的混合进料的使用似乎减轻了蛋白质生产带来的代谢负担，从而提高了比生产率和体积生产率。但是，通过使用〜（13）C衍生的代谢通量比（METAFoR）约束条件通过经典代谢通量分析对巴斯德毕赤酵母中甘油/甲醇共代谢的初步研究受到了使用C3：C1底物混合物时减少的标记信息的阻碍。关于常规的C6底物，即葡萄糖。在这项研究中，使用生物合成定向分数〜（13）C标记进一步表征了甘油/甲醇共混化条件下通过中心代谢途径的碳通量分布。特别是，通过使用软件工具〜（13）CFlux2迭代拟合来自蛋白氨基酸的NMR衍生的13C标记数据，在3种不同的甘油/甲醇比和生长速率下获得了代谢通量分布。具体而言，将细胞在需氧量为80：20、60：40和40:60（w：w）的甘油/甲醇混合物以两种稀释率（0.05hour〜（-1）和0.16hour〜（-1） ）），从而可以获取其他数据（生物量组成和细胞外通量）以补充现有数据集。进行的〜（13）C-MFA结果表明，稀释率发生了变化，中心碳代谢中的碳通量发生了显着的重新分配，而碳源的比例对生长在60°C的细胞中的碳通量分布的影响较小。相同的稀释率。在低增长率下，直接与CO_2同化的甲醇百分比在50％至70％之间。在高增长率下，在两个甲醇含量最高的条件下，甲醇通过直接途径完全被CO 2吸收。

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