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《Nature》:DNA编辑首次实现“硼-碳键”生物制造

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来源|科学网博客

加州理工大学化学工程、生物工程和生物化学教授Frances Arnold和她的团队独创了一种能生产硼-碳键的细菌。硼-碳键只能通过化学人工合成，从来没听说过天然有机生物能生产这种化学键的。

在合成生物学领域，各种生命被科学家们随手拿来制造药品、农药、甚至“智能小机器人”。只有想不到没有做不到。去年，Arnold的研究小组报道生产硅-碳键的细菌发表了一篇《Science》，今年11月29日，他们又拿出了生产硼-碳键的细菌发表了一篇《Nature》。

分子中含有硅-碳键的有机化合物被称为有机硅化合物，在工业界具有广泛用途。硼与一些有机基团生成的有机化合物被称为有机硼化合物，除了在有机合成方面应用广泛以外，有机硼化合物还可用作聚合反应的引发剂、煤油抗氧化剂、肥料、杀菌剂和抗癌药等。

“用生物代替化工生产更环保、更经济、中间产物毒性更小，”Arnold说。

“这次，前所未有的，我们赋予生命体生产一种新化学键的能力，”Arnold说。“但这仅仅是个开始，我们将开辟一个全新的发明创造探索空间。”

人类正逐渐将改造自然和利用自然的本领发挥到极致。

早在1990年代，Arnold就开发了一种被称为定向进化（directed evolution）的方法，诱使细菌制造含硼化合物。他们让酶在实验室中进行进化以完成预期功能，以生产自然生物界中不存在的化学键。

科学家们从一种由热泉细菌生产的细胞色素c（cytochrome c）变异体蛋白开始，突变了编码该蛋白的DNA，然后把突变DNA序列放入上千个细菌细胞内，看哪些细菌能生成所需的硼-碳键。筛选出的突变DNA再经过数次突变-检测循环，直到细菌们高度熟练硼-碳化合物组装。

他们总共制造了6个版本的蛋白，每种都能自如运用硼-碳键组装分子，但是6种分子的结构稍有不同。研究结果证明，在生产原理完全一致的情况下，细菌们的目标产出率比合成化工法高400倍。

“蛋白质DNA就像软件，能黑客也能被重写，”文章一作Jennifer Kan说。“传统化学，如果你想生产某个新东西，你必须重新合成一整套化学催化剂。但是合成生物不同，你只需修改DNA，告诉细菌你想要的东西长什么样。”

“硼是化学界的无名英雄之一，虽然它不经常被人挂在嘴边，但它对化学界的贡献非常巨大。最近美国宇航局的好奇漫游者在火星上发现了硼元素，这是一颗星球可能适合居住的标志性条件之一。我们很高兴，成为率先把硼元素放进合成生物工具箱的人。”

Genetically programmed chiral organoborane synthesis

Recent advances in enzyme engineering and design have expanded nature’s catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon–boron bonds in the presence of borane–Lewis base complexes, through carbene insertion into boron–hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h–1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.