Acyl Carrier Proteins: The key to successfully engineering new biosynthetic pathways.

聚酮化合物天然产物（由聚酮化合物水解酶（PKS）产生）目前作为高价值化合物的巨大来源，其应用范围包括农用化学品、抗生素、抗癌和人类和兽医学以及用于生物技术研究的工具化合物。在理解PKS的化学、生物化学和结构生物学方面已经取得了巨大的进步，PKS是具有多种结构的酶的复杂集合。例如，在假单胞菌酸A（莫匹罗星的主要成分）的生物合成中，64种不同的酶，其中一些被组织成共价连接的子集，而其他的是独立的。天然产物经由衍生自CoA（例如丙二酰-CoA）的简单碳结构单元的进化可以在由共价连接的模块中的酶的线性排列决定的系统性顺式途径中进行，但关键地可以在关键接合点处募集独立的反式配偶体以进行额外的化学反应。这些组装体中最常见的蛋白质是酰基载体蛋白（ACP），它控制中间体从PKS的一个催化位点到下一个催化位点的传递（图1），并且还可能编码在需要时可以招募反式伴侣的基序。它们也可以排列为二-甚至三-ACP重复序列，这为它们的行为增加了额外的复杂性。这些蛋白质是这些PKS的核心，但底物特异性和蛋白质-蛋白质相互作用等因素仍然知之甚少，但仍然是成功操纵这些途径以生产高价值化学品的关键。我们是国际公认的该领域的领导者，最近我们使用结合化学、生物信息学、分子生物学和核磁共振的多学科方法，提出了一套规则，指导ACP如何专门招募一组反式作用酶，以专门掺入b分支莫匹罗星分子组装的关键阶段（Haines等人，（2013）9，685 - 692）。我们将这些研究扩展到新的ACP的结构生物学，包括三结构域ACP，以了解这些较大的组件如何发挥作用，以及它们如何特异性地招募其他反式作用伙伴（例如烯酰还原酶）。这将需要合成化学家（CLW），X射线晶体学（PRR），NMR光谱学（MPC）以及生物信息学（见合作者）的技能和专业知识。我们将从莫匹罗星和卡利曼他辛途径中提取例子，这两种途径都包含这些ACP的例子，每个途径多达16种不同的ACP，它们提供了模型，独立的正交集，用于测试重要的识别控制点。

Polyketide natural products (produced by polyketide synthases (PKSs)) currently serve as a vast source of high value compounds with applications spanning agrochemicals, antibiotics, anti-cancer and human and vetinary medicine as well as tool compounds for use in biotechnological research. Enormous strides have been made in understanding the chemistry, biochemistry and structural biology of the PKSs that are complex collections of enzymes with a diverse array of architectures. For example in the biosynthesis of pseudomonic acid A, the main component of mupirocin, 64 different enzymes, some of which are organised into covalently linked subsets and others are free-standing. The evolution of the natural product via simple carbon building blocks derived from CoA (e.g. malonyl-CoA) can progress in a systematic, in-cis, pathway dictated by the linear arrangement of enzymes in the covalently linked modules but critically can recruit free-standing in-trans partners at critical junctures to perform additional chemistry. The most common protein within these assemblies, the acyl carrier protein (ACP), controls the passage of intermediates from one catalytic site of the PKS to the next (Figure 1) and may also encode motifs that can recruit trans-partners when required. They can also be arranged as di- or even tri-ACP repeats that adds an additional layer of complexity to their behaviour. These proteins are central to these PKSs but factors such as substrate specificity and protein-protein interactions remain poorly understood yet hold the key to successfully manipulating these pathways to produce high-value chemicals. We are internationally recognized as leaders in this field and recently we have used a multi-disciplinary approach combining chemistry, bioinformatics, molecular biology and NMR to propose a set of rules governing how ACPs specifically recruit a set of trans-acting enzymes to specifically incorporate b-branches at key stages of the molecular assembly of mupirocin (Haines et al., Nat. Chem. Biol. (2013) 9, 685-692). We will extend these studies to the structural biology of new ACPs, including tri-domain ACPs, to understand how these larger assemblies function and how they specifically recruit other trans-acting partners (e.g. enoyl-reductases). This will require the skills and expertise of both synthetic chemists (CLW), X-ray crystallography (PRR), NMR Spectroscopy (MPC) as well as bioinformatics (see collaborators). We will draw examples from the mupirocin and kalimantacin pathways, both of which contain examples of these ACPs and with up to 16 different ACPs per pathway, they provide model, self-contained orthogonal sets with which to test vital recognition control points.