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

 描述(由申请人提供) 聚羟基烷酸酯(PHAs)是由除碳以外的多种细菌在营养有限的生长条件下产生的聚氧酯。由于其良好的生物相容性、生物降解性和多功能性，PHA已被开发用于医疗器械、药物输送和组织工程的各种生物医学应用。FDA在2009年批准了PHAs的第一次医疗用途，作为一种可吸收缝合线，商标为TephaFLEX。然而，PHA的生产成本过高一直是其进一步发展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，以便能够经济地生产具有特定性质的PHA。为了促进这一点，本提案将重点放在PHA合成酶(PHAC)和Phasin蛋白(PHAP)上，这两个蛋白对PHA的生产和所生产的材料的性质都是关键。具体目的是：(1)研究PHAC在PHA生产中的作用机理和分子量的控制。我们将使用涉及酶学、分子生物学和合成化学的多种方法来研究I类合成酶的链延长，这些合成酶比III类酶更具挑战性。还将努力寻找建议参与控制使用转基因生物的PHA MWs的“额外因素”。蛋白质-蛋白质的相互作用将通过强相互作用的下拉试验和弱相互作用的光活性非天然氨基酸来识别。PHAC本身对分子量的控制也将通过合成类似物在体外或在体内通过鉴定链终止/重新引发过程中涉及的残基来研究；(2)通过X射线结晶学获得PHA合成酶的结构信息。Geisbrecht博士是同一所大学的结晶学家，在他的合作下，来自不同细菌来源的合酶将被提纯，并在没有和存在配体的情况下进行结晶筛选。我们与不可水解性CoA类似物共结晶的初步结果为初步形成PHAC结构提供了一条明确的途径。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，并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通过各种结合分析和添加PHA生物合成途径的大肠杆菌来表征。通过荧光显微镜和点击化学的结合，将首次在体内监测颗粒的形成。在分子水平上阐明PHAc、Phap、“附加因子”和颗粒(PHA)形成的作用和关系，对于我们更好地理解PHA的产生具有重要意义。最终，这将允许具有特定性质的PHA被经济地生产用于医疗应用。我们的结果也将揭示模板无关聚合的广泛反应，其中的机制仍然是谜。

 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.