Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.
Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway.
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DOI：
10.1186/1475-2859-11-22
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发表时间：
2012-02-13
影响因子：
6.4
通讯作者：
Glieder A
中科院分区：
文献类型：
--
作者：
Krainer FW;Dietzsch C;Hajek T;Herwig C;Spadiut O;Glieder A
甲醇营养型酵母毕赤酵母（Pichia pastoris）已成为重组蛋白生产的重要宿主生物，并且能够使用甲醇作为唯一碳源。甲醇利用途径描述了甲醇代谢过程中发生的所有催化反应。尽管某些关键酶在该途径中的重要性，但到目前为止，关于过表达这些关键酶中的任一种对巴斯德毕赤酵母菌株中的总体能量行为、生产率和底物摄取速率的可能影响知之甚少。采用一种基于甲醇脉冲分批培养的快速简便方法来表征不同的巴斯德毕赤酵母菌株。发现具有MutS表型的菌株在体积生产率和表达重组辣根过氧化物酶C1 A的效率方面均优于具有Mut+表型的菌株上级。因此，在甲醇利用途径中发挥关键作用的二羟丙酮合成酶、转酮酶或甲醛脱氢酶中的任何一种在含有报告酶辣根过氧化物酶或南极假丝酵母脂肪酶B的MutS菌株中共过表达。虽然这些酶的共过表达没有改变重组MutS菌株的化学计量产率，但观察到比生长速率、比底物摄取速率和比生产率的显著变化。二羟丙酮合酶的共过表达产生了2- 3倍更有效的底物甲醇转化为产物，但也导致了降低的体积生产率。甲醛脱氢酶的共过表达导致底物转化为产物的效率提高2倍，并且与没有工程化甲醇利用途径的菌株相比至少具有相似的体积生产率，因此被证明是改善重组蛋白生产的有价值的策略。共过表达甲醇利用途径相关酶显著影响重组毕赤酵母MutS菌株的比生长速率、甲醇摄取和比生产力。最近开发的一种方法，以确定菌株的具体参数的基础上动态分批培养被证明是一个有价值的工具，快速应变表征，从而早期的工艺开发。
The methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains. A fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production. Co-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.
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影响因子：
14.9
作者：
Hartner FS;Ruth C;Langenegger D;Johnson SN;Hyka P;Lin-Cereghino GP;Lin-Cereghino J;Kovar K;Cregg JM;Glieder A
通讯作者：
Glieder A
影响因子：
2.8
作者：
Lee, B;Yurimoto, H;Kato, N
通讯作者：
Kato, N
DOI：
10.1038/nbt0587-479
发表时间：
1987-05-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
CREGG, JM;TSCHOPP, JF;THILL, GP
通讯作者：
THILL, GP
影响因子：
4.7
作者：
Abad, Sandra;Kitz, Kerstin;Glieder, Anton
通讯作者：
Glieder, Anton
DOI：
10.1093/protein/13.5.377
发表时间：
2000-05-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Morawski, B;Lin, ZL;Arnold, FH
通讯作者：
Arnold, FH