Use of a mixture of glucose and methanol as substrates for the production of recombinant trypsinogen in continuous cultures with Pichia pastoris Mut+
Use of a mixture of glucose and methanol as substrates for the production of recombinant trypsinogen in continuous cultures with Pichia pastoris Mut+
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DOI：
10.1016/j.jbiotec.2011.10.010
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发表时间：
2012-01-01
影响因子：
4.1
通讯作者：
Kovar, Karin
中科院分区：
文献类型：
--
作者：
Paulova, Leona;Hyka, Petr;Kovar, Karin
纯甲醇是毕赤酵母重组蛋白生产过程中 AOX1 启动子的诱导剂和碳/能源所必需的，但它是不切实际的，因此通常是不受欢迎的。作为替代方案，检查了使用双碳底物的程序（11.7g（碳）l(-1)，来自葡萄糖/甲醇的60%/40%碳）。分析了对甲醇代谢、猪胰蛋白酶原细胞外形成、生物量生长和细胞活力的影响。与连续利用葡萄糖和甲醇的分批培养相反，在碳/能量限制的连续培养中（在稀释率0.03和0.20 h(-1)之间操作），葡萄糖对甲醇利用的抑制作用在0.15 h(-1)内被消除（约130%的mu(max)与甲醇）。使用该混合物，发现生物质的产量（1.54垂直于0.12）g（CDW）g（碳）（-1）比单独使用甲醇的产量大1.4倍。尽管目前普遍认为葡萄糖对 AOX1 启动子具有抑制作用，但该产品是在整个稀释率范围内合成的，在 0.07 h(-1) 时的最大生产率为 (0.70 +/- 0.12) mg(CDW)g(-1) h(-1)。因此，在毕赤酵母Mut(+)菌株的重组蛋白生产中，葡萄糖被证明是甲醇的可行的部分替代品，同时提高了工艺生产率。 (C) 2011 Elsevier B.V. 保留所有权利。
Pure methanol, which is required as an inducer of the AOX1 promoter and a carbon/energy source in processes for recombinant protein production by Pichia pastoris, is impracticable and therefore generally undesirable. As an alternative, a procedure using double carbon substrate was examined (11.7 g(carbon) l(-1), 60%/40% carbon from glucose/methanol). The effects on methanol metabolism, extracellular formation of porcine trypsinogen, biomass growth and cell viability were analyzed. In contrast to batch cultures, where the glucose and methanol were utilized sequentially, in carbon/energy-limited continuous cultures (operated between dilution rates 0.03 and 0.20 h(-1)) the repressive effect of glucose on methanol utilization was eliminated up to 0.15 h(-1) (ca. 130% of mu(max) with methanol). With the mixture, the yield of biomass (1.54 perpendicular to 0.12) g(CDW) g(carbon)(-1) was found to be 1.4 times larger than the yield with methanol alone. Despite the current widespread view that glucose has a repressive effect on the AOX1 promoter, the product was synthesized over the entire range of dilution rates, with maximum productivities of (0.70 +/- 0.12) mg(CDW)g(-1) h(-1) at 0.07 h(-1). Thus, glucose was shown to be a feasible partial substitute for methanol in recombinant protein production by P. pastoris Mut(+) strain while enhancing process productivity. (C) 2011 Elsevier B.V. All rights reserved.