将非典型氨基酸（ncAAs）引入到蛋白质中已被证明是调控蛋白质结构和功能以及研究许多生物学过程的有效手段。提高含ncAA蛋白的产率具有重要的工业化应用意义。大肠杆菌C321 ΔA是通过替换基因组中所有已知的琥珀密码子和删除RF1而产生的，已被证明是使用遗传密码技术扩展表达含ncAA蛋白质的理想宿主。
2021 年 9 月，西南医科大学高晓威团队在Frontiers in Microbiology发表题为“Comparative Analyses of the Transcriptome and Proteome of Escherichia coli C321.ΔA and Further Improving Its Noncanonical Amino Acids Containing Protein Expression Ability by Integration of T7 RNA Polymerase”的文章。
考虑到全基因组的密码子重组可能会影响基因的转录和翻译，该研究比较分析了重组菌株大肠杆菌C321.ΔA的转录组和蛋白质组。结果显示与它的母株EcNR2相比，大肠杆菌C321.ΔA一些功能和代谢途径有不同的表达，参与碳水化合物和能量代谢的基因被明显地下调了。该结果可能为解释大肠杆菌C321.ΔA的生长缺陷提供了重要线索。
由于T7 RNA聚合酶进行DNA转录的速度大约是大肠杆菌RNA聚合酶的五倍，T7 RNA聚合酶系统已经成为大肠杆菌中最广泛使用的表达系统之一。为了能够在大肠杆菌C321.ΔA. exp中使用pET系统表达含有ncAAs的蛋白质，并进一步提高含ncAA的蛋白质的产量，该研究尝试使用CRISPR-Cas9系统将T7 RNA聚合酶基因整合进基因组（图1）。
图1 将T7 RNA聚合酶整合进入大肠杆菌C321.ΔA.exp基因组的策略
通过使用pET系列质粒进行重组蛋白表达，研究了插入的T7 RNA聚合酶基因是否能在大肠杆菌C321.ΔA. exp中工作。如图2所示，大肠杆菌C321.ΔA.exp和大肠杆菌C321.ΔA.exp T7在用阿拉伯糖诱导后携带pBAD24-GFP质粒时都能检测到强烈的GFP荧光。然而，当两株菌株都带有pET26b-GFP质粒时，只有大肠杆菌C321.ΔA.exp T7在用IPTG诱导后显示出明显的GFP荧光，这是因为缺乏T7 RNA聚合酶，pET质粒不能在大肠杆菌C321.ΔA.exp中发挥功能，但可以在大肠杆菌C321.ΔA.exp T7中很好地工作。
图2 GFP在大肠杆菌C321.ΔA exp和大肠杆菌C321.ΔA.exp T7中的表达
为了测试在大肠杆菌C321.ΔA.exp T7的多个UAG位点上整合ncAAs的能力，研究人员使用两个广泛报道的正交质粒（pEVOL和pUltra衍生物）将多个ncAAs整合到GFP中。
图3 不同菌株中pIoF与含有琥珀密码子突变的GFP的结合
如图3A所示，当使用pET26b作为表达GFP Y151TAG基因的载体时，只有大肠杆菌BL21（DE3）和大肠杆菌C321.ΔA.exp T7菌株在补充1 mM pIoF时有强烈的细胞内GFP荧光。大肠杆菌DH10B和大肠杆菌C321.ΔA.exp菌株由于缺乏T7 RNA聚合酶和不能使用pET26质粒中的T7启动子而没有显示任何可见的荧光。当使用质粒pBAD24作为表达GFP Y151TAG基因的载体时，所有四个菌株在1 mM pIoF存在的情况下都显示出显著的细胞内GFP荧光。大肠杆菌DH10B比大肠杆菌BL21（DE3）显示出更强的荧光，这可能是由于大肠杆菌DH10B不会分解诱导剂L-阿拉伯糖，可以表达更多完整的含pIoF的GFP突变体。事实证明，大肠杆菌C321.ΔA及其衍生菌株作为表达宿主可以显著提高含ncAA蛋白的产量，尤其是针对在多个位点含有nsAAs的表达蛋白（图3B）。
在这项研究中，研究人员分析了大肠杆菌C321.△A的转录组和蛋白质组概况，发现了一些差异表达的功能和代谢途径。这些结果表明，RF1的缺失和/或UAG密码子的重新分配可能削弱了大肠杆菌的碳水化合物利用和能量生成，这可能是大肠杆菌C321.ΔA表现出生长缺陷的原因之一。此外，该研究通过整合T7 RNA聚合酶系统，提高了大肠杆菌C321.ΔA中含ncAA蛋白的产量。
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