Lipinszki Zoltan, Vernyik Viktor, Farago Nora, Sari Tobias, Puskas Laszlo G, Blattner Frederick R, Posfai Gyorgy, Gyorfy Zsuzsanna
Scarab Genomics LLC, Madison , Wisconsin 53713 , United States.
ACS Synth Biol. 2018 Nov 16;7(11):2656-2664. doi: 10.1021/acssynbio.8b00332. Epub 2018 Nov 2.
Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.
大肠杆菌是一种成熟且常用的蛋白质异源表达宿主。然而，宿主与重组蛋白的原始来源在同义密码子选择上的偏好（密码子偏性）可能不同，这构成了生产中的一个潜在瓶颈。密码子选择通过一种复杂且了解甚少的机制影响翻译效率。某些tRNA种类的可用性是可能限制翻译能力的因素之一。在此，我们提供了一种过表达tRNA的策略，该策略能够解决密码子偏性问题，并在遇到稀有密码子时提高常用宿主BL21(DE3)的翻译能力。在一种名为SixPack的源自BL21(DE3)的菌株中，对应于六种丰度最低的tRNA种类的基因拷贝被组装在一个合成片段中，并插入到一个rRNA操纵子中。这种排列方式在不干扰新菌株生长特性的同时，允许对额外tRNA基因的转录进行动态控制，从而在指数生长期显著提高稀有tRNA的水平。对一组具有不同来源和密码子组成的重组蛋白进行表达分析的结果表明，SixPack的表现优于亲本BL21(DE3)或配备了表达稀有tRNA质粒的相关菌株。
