Molecular Study of PHA Biosynthesis: Production of Biodegradable Polymers for Medical Applications

 描述（由申请人提供） 聚羟基链烷酸酯（PHA）是由多种细菌在除碳之外的营养物受限的生长条件下产生的聚氧酸酯。由于其优异的生物相容性、生物降解性和多功能性，PHA已被开发用于医疗器械、药物递送和组织工程中的各种生物医学应用。FDA于2009年批准PHA作为可吸收缝线的首次医疗用途，商品名为TephaFLEX。然而，PHA生产的高成本一直是其进一步开发和下游商业化的障碍。我们的目标是识别和了解完整的PHA生物合成机制，以便可以经济地生产具有特定特性的PHA。为了促进这一点，本提案将集中在PHA合成酶（PhaC）和相蛋白（PhaP），这是关键的PHA生产和所产生的材料的性质。具体目的是：（1）表征PhaC在PHA生产中的机理和分子量（MW）的控制。我们将研究链延长的I类内切酶是更具挑战性的III类酶使用多种方法，涉及酶学，分子生物学和合成化学。还将努力寻找拟参与使用转基因生物控制PHA MW的“其他因素”。蛋白质-蛋白质相互作用将通过强相互作用的下拉测定和通过掺入光活性非天然氨基酸的弱相互作用来鉴定。还将通过合成类似物在体外或通过鉴定链终止/再起始过程中涉及的残基在体内研究PhaC本身对MW的控制;（2）通过X射线晶体学获得PHA脱氢酶的结构信息。Geisbrecht博士是同一个校区的一位有成就的晶体学家，他与Geisbrecht博士合作，将对来自不同细菌来源的酶进行纯化，并在不存在和存在配体的情况下进行结晶筛选。我们与不可水解的CoA类似物共结晶的初步结果为初始PhaC结构提供了明确的途径。这种X射线结构的可用性将为我们提供关于底物识别和酶机制的有价值的见解，以及使我们能够实现蛋白质工程的长期目标;（3）表征PhaP在PHA生产和颗粒形成中的作用。PhaC和PhaP的关系将在体外和体内使用各种结合试验和补充PHA生物合成途径的大肠杆菌进行表征。将通过荧光显微镜和点击化学的组合首次在体内监测颗粒形成。从分子水平阐明PhaC、PhaP、“附加因子”和颗粒（PHA）形成的作用和关系对于完善我们对PHA生产的理解具有重要意义。最终，这将允许具有定义属性的PHA经济地生产用于医疗应用。我们的研究结果也将揭示广泛的反应的模板无关的聚合反应的机制仍然是谜。

 DESCRIPTION (provided by applicant) Polyhydroxyalkanoates (PHAs) are polyoxoesters produced by a wide range of bacteria under nutrient-limited growth conditions except for carbon. Due to their excellent biocompatibility, biodegradability, and versatility, PHAs have been developed for various biomedical applications in medical devices, drug delivery, and tissue engineering. The FDA approved the first medical use of PHAs in 2009 as an absorbable suture under the trade name TephaFLEX. However, the high cost of PHA production has been an impediment to their further development and downstream commercialization. Our goal is to identify and understand the complete PHA biosynthetic machinery so that PHAs with defined properties can be produced economically. To facilitate this, the present proposal will focus on the PHA synthase (PhaC) and phasin protein (PhaP), which are key to both PHA production and the properties of the material produced. The specific aims are: (1) to characterize the mechanism of PhaC in PHA production and control of molecular weight (MW). We will investigate chain elongation of class I synthases that are much more challenging than the class III enzymes using multiple approaches involving enzymology, molecular biology, and synthetic chemistry. Efforts will also be made to look for the "additional factors" that are proposed to participate in the control of PHA MWs using genetically modified organisms. Protein-protein interactions will be identified through pull-down assays for strong interactions and by incorporating photoactive unnatural amino acids for weak interactions. The MW control by PhaC itself will also be studied in vitro through a synthetic analog or in vivo through identifying the residues involved in the chain termination/re-initiation processes; (2) to obtain structural information on PHA synthases through X-ray crystallography. In collaboration with Dr. Geisbrecht who is an accomplished crystallographer on the same campus, synthases from different bacterial sources will be purified and screened for crystallization in the absence and presence of ligands. Our preliminary results of co-crystallization with a nonhydrolyzable CoA analog have provided a clear path toward an initial PhaC structure. The availability of this X-ray structure will provide us with valuable insight on substrate recognition and enzyme mechanism as well as enabling our long- term goal of protein engineering; (3) to characterize roles of PhaP in PHA production and granule formation. The relationship of PhaC and PhaP will be characterized in vitro and in vivo using various binding assays and with Escherichia coli supplemented with a PHA biosynthetic pathway. Granule formation will be monitored in vivo for the first time through a combination of fluorescence microscopy and click-chemistry. Elucidating the roles and relationships of PhaC, PhaP, "additional factors" and granule (PHA) formation at the molecular level is of great importance to complete our understanding of PHA production. Ultimately, this will allow PHAs with defined properties to be economically produced for medical applications. Our results will also shed light on the widespread reactions of template-independent polymerizations where the mechanism remains enigmatic.