1] Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee, USA. [2].
Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, Tennessee, USA.
Nat Chem Biol. 2014 May;10(5):392-9. doi: 10.1038/nchembio.1494. Epub 2014 Mar 23.
Concatenation of engineered biocatalysts into multistep pathways markedly increases their utility, but the development of generalizable assembly methods remains a major challenge. Herein we evaluate 'bioretrosynthesis', which is an application of the retrograde evolution hypothesis, for biosynthetic pathway construction. To test bioretrosynthesis, we engineered a pathway for synthesis of the antiretroviral nucleoside analog didanosine (2',3'-dideoxyinosine). Applying both directed evolution- and structure-based approaches, we began pathway construction with a retro-extension from an engineered purine nucleoside phosphorylase and evolved 1,5-phosphopentomutase to accept the substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate selectivity and threefold increased turnover in cell lysate. A subsequent retrograde pathway extension, via ribokinase engineering, resulted in a didanosine pathway with a 9,500-fold change in nucleoside production selectivity and 50-fold increase in didanosine production. Unexpectedly, the result of this bioretrosynthetic step was not a retro-extension from phosphopentomutase but rather the discovery of a fortuitous pathway-shortening bypass via the engineered ribokinase.
将工程化的生物催化剂串联成多步途径显著提高了它们的效用，但开发通用的组装方法仍然是一个主要挑战。本文我们评估了“生物回溯合成”，这是逆行进化假说的一个应用，用于合成途径的构建。为了测试生物回溯合成，我们设计了一条合成抗逆转录病毒核苷类似物双脱氧肌苷（2',3'-二脱氧肌苷）的途径。通过定向进化和基于结构的方法，我们从工程嘌呤核苷磷酸化酶开始进行反向延伸，并进化了 1,5-磷酸戊糖变位酶，以接受底物 2,3-二脱氧核糖 5-磷酸，其底物选择性提高了 700 倍，在细胞裂解物中的周转率提高了三倍。随后通过核糖激酶工程进行反向途径延伸，导致双脱氧肌苷途径的核苷产生选择性提高了 9500 倍，双脱氧肌苷产量提高了 50 倍。出乎意料的是，这个生物回溯合成步骤的结果不是从磷酸戊糖变位酶的反向延伸，而是通过工程核糖激酶偶然发现了一条捷径。
