Quantitative Physiology of Pichia pastoris During Glucose-Limited High-Cell Density Fed-Batch Cultivation for Recombinant Protein Production
Quantitative Physiology of Pichia pastoris During Glucose-Limited High-Cell Density Fed-Batch Cultivation for Recombinant Protein Production
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DOI：
10.1002/bit.22836
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发表时间：
2010-10-01
影响因子：
3.8
通讯作者：
Schmid, Andreas
中科院分区：
文献类型：
--
作者：
Heyland, Jan;Fu, Jianan;Schmid, Andreas
近年来，巴斯德毕赤酵母已成为生产蛋白质的主要微生物之一。这种发展主要是由容易获得的遗传工具和容易使用甲醇（或甲醇/甘油混合物）作为诱导剂和碳源的高细胞密度培养驱动的。为了克服所观察到的甲醇使用的局限性，例如高热显影、细胞列表和爆炸危险，我们在这里重新审视了使用葡萄糖作为唯一碳源用巴斯德毕赤酵母生产蛋白质的可能性。在葡萄糖限制的补料分批培养中使用重组巴斯德毕赤酵母菌株，达到非常高的湿细胞密度（超过200 g（CDW）L-1），导致约6.5 g L-1的重组蛋白滴度。为了研究重组蛋白生产和高细胞密度发酵对巴斯德毕赤酵母代谢的影响，我们在分批和分批补料实验中使用基于C-13-示踪剂的代谢通量分析。在补料分批实验中，在0.12 h（-1）的受控生长速率下，与重组菌株和参考菌株的0.7 mmol g（-1）h（-1）相比，TCA循环通量增加了1.1 mmol g（-1）h（-1），这表明碳和能量资源的通量重新路由有限但显著。这种通量的变化很可能是蛋白质合成的原因。总之，结果突出了葡萄糖作为碳源和能源的潜力，从而实现了高生物质浓度和蛋白质滴度。对重组蛋白生产过程中代谢过程的深入了解将对菌株设计和发酵开发具有指导意义。Biotechnol. Bioeng. 2010，107：357-368（c）2010 Wiley Periodicals，Inc
Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high-cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lists, and explosion hazard, we here revisited the possibility to produce proteins with P pastoris using glucose as sole carbon source. Using a recombinant P pastoris strain in glucose limited fed-batch cultivations, very high-cell densities wet e reached (more than 200 g(CDW) L-1) resulting in a recombinant protein titer of about 6.5 g L-1. To investigate the impact of recombinant protein production and high-cell density fermentation on the metabolism of P pastoris, we used C-13-tracer-based metabolic flux analysis in batch and fed-batch experiments. At a controlled growth rate of 0.12 h(-1) in fed-batch experiments an increased TCA cycle flux of 1.1 mmol g(-1) h(-1) compared to 0.7 mmol g(-1) h(-1) for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010,107: 357-368 (c) 2010 Wiley Periodicals, Inc