Efficient rational modification of non-ribosomal peptides by adenylation domain substitution
Efficient rational modification of non-ribosomal peptides by adenylation domain substitution
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DOI：
10.1038/s41467-020-18365-0
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发表时间：
2020-09-11
影响因子：
16.6
通讯作者：
Ackerley, David F.
中科院分区：
文献类型：
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作者：
Calcott, Mark J.;Owen, Jeremy G.;Ackerley, David F.
非核糖体肽合成酶（NRPS）形成模块化装配线，其中每个模块控制特定单体并入短肽产物中。模块由一个或多个关键结构域组成，包括腺苷酸化（A）结构域，其识别并活化单体底物;缩合（C）结构域，其催化酰胺键形成;和硫醇化（T）结构域，其在催化结构域之间穿梭反应中间体。这种安排提供了合理的肽修饰的前景，通过取代基板指定的结构域。20多年来，人们一直认为C结构域在校对底物中起着关键作用;这一假设使理性NRPS重新设计变得非常复杂。在这里，我们提出的证据，从定向和自然进化的研究表明，任何基板指定的C结构域的作用很可能是例外，而不是规则，和新的非核糖体肽可以产生单独的A结构域的取代。我们确定了允许的A结构域重组边界，并表明这些允许我们以高产率有效地产生修饰的绿脓菌荧光素肽。我们进一步证明了我们的方法在最初用于推断C结构域底物特异性的PheATE-ProCAT模型系统中的可转移性，以与现行教条不一致的产率产生修饰的二肽产物。
Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma.