Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint.
Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint.
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DOI：
10.1186/1475-2859-8-27
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发表时间：
2009-05-22
影响因子：
6.4
通讯作者：
Domach MM
中科院分区：
文献类型：
--
作者：
Cunningham DS;Koepsel RR;Ataai MM;Domach MM
质粒被重新考虑作为基因治疗和疫苗的病毒的可行载体替代品，因为它们更安全，无毒，更容易生产。因此，人们重新关注作为生物过程的治疗性终产物的质粒DNA本身的生产。提高此类新兴工艺的最佳当前产率和生产率将有助于确保工业规模的经济可行性。因此，我们的目标是建立大肠杆菌代谢的化学计量模型，以（1）确定其最大理论质粒生产能力，（2）确定显着影响质粒生产的因素。这种模型被开发用于在葡萄糖基本培养基上分批需氧生长的条件下生产高拷贝质粒。该模型的目标是使质粒产量最大化。通过采用某些约束条件并检查所得通量分布，确定了显著影响质粒产率的几个因素。乙酸产生和质粒的抗生素抗性标记的组成型表达产生负面影响，而低丙酮酸激酶（Pyk）通量和通过转氢酶活性产生NADPH提供正面影响。最高的理论产量（592毫克/克）的条件下，没有标记或乙酸生产，零Pyk流量，和最大允许的转氢酶活性。为了比较，当这四种通量被限制为野生型值时，产生几十mg/g的数量级的产率，这与迄今报道的最佳实验产率相当。这些结果表明，特定的质粒产量理论上可以达到目前实验最大值（51 mg/g）的12倍。此外，它们意味着当设计用于质粒生产的宿主菌株时，消除Pyk活性和/或转氢酶上调将是实施的有用策略;减少乙酸盐生产的突变也将是有利的。结果进一步表明，使用一些其他方法进行质粒选择而不是抗生素抗性，或者至少削弱标记物的表达，将是有益的，因为它将允许更多的前体代谢物，能量和还原力用于质粒生产。到目前为止，已经通过实验探索了消除Pyk活性的影响，结果是质粒产量显著提高。
Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of Escherichia coli metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production. Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date. These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the marker's expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.
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影响因子：
3.8
作者：
Flores, S;de Anda-Herrera, R;Bolívar, FG
通讯作者：
Bolívar, FG
影响因子：
3.2
作者：
Danquah, Michael K.;Forde, Gareth M.
通讯作者：
Forde, Gareth M.
影响因子：
3.4
作者：
Ow, Dave Siak-Wei;Nissom, Peter Morin;Yap, Miranda Gek-Sim
通讯作者：
Yap, Miranda Gek-Sim
影响因子：
4.1
作者：
Hägg, P;de Pohl, JW;Isaksson, LA
通讯作者：
Isaksson, LA
DOI：
10.1007/bf00330395
发表时间：
1986-01-01
期刊：
MOLECULAR AND GENERAL GENETICS
影响因子：
--
作者：
LINCHAO, S;BREMER, H
通讯作者：
BREMER, H