Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates.
Pichia pastoris Exhibits High Viability and a Low Maintenance Energy Requirement at Near-Zero Specific Growth Rates.
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DOI：
10.1128/aem.00638-16
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发表时间：
2016-08-01
影响因子：
4.4
通讯作者：
Mattanovich D
中科院分区：
文献类型：
--
作者：
Rebnegger C;Vos T;Graf AB;Valli M;Pronk JT;Daran-Lapujade P;Mattanovich D
巴斯德毕赤酵母是生产重组蛋白的广泛应用宿主。在极低的生长速度下了解其生理特性，是将产品形成与细胞生长脱钩的第一步，因此具有生物技术意义。在极低的特定生长率下，Retentostat培养是研究微生物的一种很好的工具，但到目前为止还没有在巴斯德毕赤酵母中实施。根据稳态葡萄糖限制恒化器培养估算的维持能量需要量(Ms)和葡萄糖的最大生物量产量(Yx/Smax)来确定摄食方案。好氧保持器培养实现了从比生长率(μ)0.025 h−1到接近零的比生长率(μ<；0.001 h−1)的可重复、平稳的过渡。在这些接近于零的特定增长率下，生存能力至少保持了97%。在接近零的生长速率下，ms的值为3.1±0.1 mg葡萄糖/g生物量和h，比快速生长的恒化器培养估计的ms低3倍。这一差异表明，巴斯德毕赤酵母在极低的μ下减少了维持能量需求，这是以前在真核生物中没有观察到的现象。在维持剂培养过程中，细胞内糖原和海藻糖水平升高，而μ水平逐渐下降。接近零增长的转录重编程包括许多转录因子和应激相关基因的上调以及细胞周期基因的下调。这项研究强调了维持性能量代谢比较分析的相关性，维持性能量代谢对大规模工业过程具有重要影响。重要：毕赤酵母天然地生活在树上，可以利用不同的碳源，其中包括葡萄糖、甘油和甲醇。在生物技术中，它被广泛用于生产重组蛋白。无论是对其自然栖息地中的生命的理解，还是对生产过程的优化，在极低的生长速度下更好地了解细胞生理学都将具有非凡的价值。因此，我们在恒温器中培养巴斯德毕赤酵母，即使在零生长的情况下也能培养出代谢活性细胞。在这里，我们达到了长达38天的倍增，并发现巴斯德毕赤酵母在非常缓慢的生长过程中将其维持能量需求减少了3倍，这使得它能够在比面包师酵母低得多的底物供应下生存。
The yeast Pichia pastoris is a widely used host for recombinant protein production. Understanding its physiology at extremely low growth rates is a first step in the direction of decoupling product formation from cellular growth and therefore of biotechnological relevance. Retentostat cultivation is an excellent tool for studying microbes at extremely low specific growth rates but has so far not been implemented for P. pastoris. Retentostat feeding regimes were based on the maintenance energy requirement (mS) and maximum biomass yield on glucose (YX/Smax) estimated from steady-state glucose-limited chemostat cultures. Aerobic retentostat cultivation enabled reproducible, smooth transitions from a specific growth rate (μ) of 0.025 h−1 to near-zero specific growth rates (μ < 0.001 h−1). At these near-zero specific growth rates, viability remained at least 97%. The value of mS at near-zero growth rates was 3.1 ± 0.1 mg glucose per g biomass and h, which was 3-fold lower than the mS estimated from faster-growing chemostat cultures. This difference indicated that P. pastoris reduces its maintenance energy requirement at extremely low μ, a phenomenon not previously observed in eukaryotes. Intracellular levels of glycogen and trehalose increased, while μ progressively declined during retentostat cultivation. Transcriptional reprogramming toward zero growth included the upregulation of many transcription factors as well as stress-related genes and the downregulation of cell cycle genes. This study underlines the relevance of comparative analysis of maintenance energy metabolism, which has an important impact on large-scale industrial processes. IMPORTANCE The yeast Pichia pastoris naturally lives on trees and can utilize different carbon sources, among them glucose, glycerol, and methanol. In biotechnology, it is widely used for the production of recombinant proteins. For both the understanding of life in its natural habitat and optimized production processes, a better understanding of cell physiology at an extremely low growth rate would be of extraordinary value. Therefore, we have grown P. pastoris in a retentostat, which allows the cultivation of metabolically active cells even at zero growth. Here we reached doubling times as long as 38 days and found that P. pastoris decreases its maintenance energy demand 3-fold during very slow growth, which enables it to survive with a much lower substrate supply than baker's yeast.