Engineering highly functional thermostable proteins using ancestral sequence reconstruction
Engineering highly functional thermostable proteins using ancestral sequence reconstruction
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DOI：
10.1038/s41929-018-0159-5
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发表时间：
2018-11-01
期刊：
影响因子：
37.8
通讯作者：
Gillam, Elizabeth M. J.
中科院分区：
文献类型：
--
作者：
Gumulya, Yosephin;Baek, Jong-Min;Gillam, Elizabeth M. J.
商业生物催化需要能够承受高温和长时间孵育的强大酶。祖先的重建表明，前寒武纪的酶往往比现存的形式更耐热。在这里，我们复活了祖先的酶，这些酶可以承受比现存形式高出30摄氏度的温度和>= 100倍的孵育时间。这在动物细胞色素P450上得到了证明，该细胞色素P450立体和区域选择性地官能化未活化的C-H键以合成有价值的化学品，以及用于制造基于丁醇的生物燃料的细菌酮醇酸还原异构酶。脊椎动物CYP 3 P450祖先的T-60（50）为66 ℃，与人类药物代谢CYP 3A 4相比，溶剂耐受性增强，但对类似的广泛底物的活性相当。祖先的酮醇酸还原异构酶在25 ℃时比同源的大肠杆菌形式高8倍，在50 ℃时增加3.5倍。因此，热稳定蛋白质可以设计使用序列数据，甚至最近的祖先。
Commercial biocatalysis requires robust enzymes that can withstand elevated temperatures and long incubations. Ancestral reconstruction has shown that pre-Cambrian enzymes were often much more thermostable than extant forms. Here, we resurrect ancestral enzymes that withstand similar to 30 degrees C higher temperatures and >= 100 times longer incubations than their extant forms. This is demonstrated on animal cytochromes P450 that stereo- and regioselectively functionalize unactivated C-H bonds for the synthesis of valuable chemicals, and bacterial ketol-acid reductoisomerases that are used to make butanol-based biofuels. The vertebrate CYP3 P450 ancestor showed a T-60(50) of 66 degrees C and enhanced solvent tolerance compared with the human drug-metabolizing CYP3A4, yet comparable activity towards a similarly broad range of substrates. The ancestral ketol-acid reductoisomerase showed an eight-fold higher specific activity than the cognate Escherichia coli form at 25 degrees C, which increased 3.5-fold at 50 degrees C. Thus, thermostable proteins can be devised using sequence data alone from even recent ancestors.