Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18/House B, A-1190 Vienna, Austria.
J Biotechnol. 2010 Apr 1;146(3):130-7. doi: 10.1016/j.jbiotec.2010.01.025. Epub 2010 Feb 6.
Plasmid DNA is being considered as a promising alternative to traditional protein vaccines or viral delivery methods for gene therapeutic applications. DNA-based products are highly flexible, stable, are easily stored and can be manufactured on a large scale. Although, much safer than viral approaches, issues have been raised with regard to safety due to possible integration of plasmid DNA into cellular DNA or spread of antibiotic resistance genes to intestinal bacteria by horizontal gene transfer. Accordingly, there is interest in methods for the production of plasmid DNA that lacks the antibiotic resistance gene to further improve their safety profile. Here, we report for the first time the gram-scale manufacturing of a minimized plasmid that is devoid of any additional sequence elements on the plasmid backbone, and merely consists of the target expression cassette and the bacterial origin of replication. Three different host/vector combinations were cultivated in a fed-batch fermentation process, comparing the progenitor strain JM108 to modified strains JM108murselect, hosting a plasmid either containing the aminoglycoside phosphotransferase which provides kanamycin resistance, or a marker-free variant of the same plasmid. The metabolic load exerted by expression of the aminoglycoside phosphotransferase was monitored by measuring ppGpp- and cAMP-levels. Moreover, we revealed that JM108 is deficient of the Lon protease and thereby refined the genotype of JM108. The main consequences of Lon-deficiency with regard to plasmid DNA production are discussed herein. Additionally, we found that the expression of the aminoglycoside phosphotransferase, conferring resistance to kanamycin, was very high in plasmid DNA producing processes that actually inclusion bodies were formed. Thereby, a severe metabolic load on the host cell was imposed, detrimental for overall plasmid yield. Hence, deleting the antibiotic resistance gene from the vector backbone is not only beneficial with regards to safety and potency of the end-product but also regarding the overall process performance.
质粒 DNA 被认为是一种很有前途的替代传统蛋白质疫苗或病毒传递方法的选择，可用于基因治疗应用。基于 DNA 的产品具有高度的灵活性、稳定性，易于储存，并且可以大规模生产。尽管与病毒方法相比安全性更高，但由于质粒 DNA 可能整合到细胞 DNA 中，或者通过水平基因转移将抗生素抗性基因传播到肠道细菌中，因此人们对其安全性提出了一些担忧。因此，人们对缺乏抗生素抗性基因的质粒 DNA 的生产方法产生了兴趣，以进一步提高其安全性。在这里，我们首次报告了在无任何额外序列元件的质粒骨架上进行大规模生产的方法，该质粒仅由目标表达盒和细菌复制起点组成。在补料分批发酵过程中，我们比较了三个不同的宿主/载体组合，包括原代菌株 JM108 和修饰菌株 JM108murselect，后者分别含有提供卡那霉素抗性的氨基糖苷磷酸转移酶的质粒，或同一质粒的无标记变体。通过测量 ppGpp 和 cAMP 水平来监测表达氨基糖苷磷酸转移酶所施加的代谢负荷。此外，我们发现 JM108 缺乏 Lon 蛋白酶，因此对 JM108 的基因型进行了改良。本文讨论了 Lon 缺陷对质粒 DNA 生产的主要影响。此外，我们发现，在形成包涵体的情况下，赋予卡那霉素抗性的氨基糖苷磷酸转移酶的表达非常高。这对宿主细胞造成了严重的代谢负担，不利于整体质粒产量。因此，从载体骨架中删除抗生素抗性基因不仅对终产物的安全性和效力有益，而且对整体工艺性能也有益。
