Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.
Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.
复制标题
DOI：
10.1371/journal.pone.0119637
复制
发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Leak DJ
中科院分区：
文献类型：
--
作者：
Edwards-Jones B;Aw R;Barton GR;Tredwell GD;Bundy JG;Leak DJ
我们已经遵循了一个典型的分批补料诱导制度下的AOX1启动子的控制下，使用微阵列和代谢组学分析的异源蛋白质生产。遗传构建体涉及1个和3个拷贝的TRY1基因，编码人胰蛋白酶原。在小规模实验室培养中，3拷贝数构建体的表达诱导了充分的未折叠蛋白反应（UPR），使得3拷贝构建体中的细胞外胰蛋白酶原滴度低于1拷贝构建体。在补料分批培养中，观察到类似的模式，其中来自1拷贝构建体的表达更高，但在这种情况下，用3拷贝菌株没有显著诱导UPR。微阵列和代谢组学信息的分析表明，3拷贝菌株在用甲醇诱导时经历细胞质氧化还原应激。在这种Crabtree阴性酵母中，这种氧化还原应激似乎延迟了对甲醇生长的适应，并抑制了异源蛋白质的产生，这可能是由于翻译中的阻断。虽然氧化还原失衡作为人为施加的缺氧的结果之前已经描述过，这是第一次，它已被表征为一个短暂的代谢失衡的结果，并显示涉及应激反应，可导致翻译停滞。如果不详细分析潜在的过程，它很容易被误解为分泌压力，通过普遍定期审议传播。
We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.
登录
查看更多内容
影响因子：
4.4
作者：
Graf A;Gasser B;Dragosits M;Sauer M;Leparc GG;Tüchler T;Kreil DP;Mattanovich D
通讯作者：
Mattanovich D
影响因子：
2.8
作者：
Inan, M;Meagher, MM
通讯作者：
Meagher, MM
影响因子：
16
作者：
Harding, HP;Novoa, I;Ron, D
通讯作者：
Ron, D
影响因子：
4.1
作者：
Jungo, Carmen;Marison, Ian;von Stockar, Urs
通讯作者：
von Stockar, Urs
影响因子：
2.8
作者：
BELLINGER, Y;LARHER, F
通讯作者：
LARHER, F