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

PHA 生物合成的分子研究：医疗应用可生物降解聚合物的生产

• 批准号: 9271549
• 负责人: Ping Li
• 金额: $ 17.5万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-05 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9271549
• 关键词: Address; Affinity Chromatography; Anabolism; Area; Attention; Bacteria; Binding; Biological Assay; Carbon; Cells; Chemistry; Clinical; Co-Immunoprecipitations; Coenzyme A; Collaborations; Crystallization; Cytoplasmic Granules; DNA; Development; Drug Delivery Systems; Engineering; Enzymatic Biochemistry; Enzymes; Escherichia coli; FDA approved; Fluorescence Microscopy; Future; Generations; Genetically Modified Organisms; Glycogen; Goals; Growth; Health; Homeostasis; Household; Human; In Vitro; Inclusion Bodies; Ligands; Light; Medical; Medical Device; Methods; Modeling; Molecular; Molecular Biology; Molecular Weight; Monitor; Names; Nature; Nutrient; Pathway interactions; Phase Transition; Polymers; Polysaccharides; Process; Production; Property; Protein Engineering; Proteins; RNA; Reaction; Regulation; Roentgen Rays; Role; Rubber; Series; Source; Starch; Structure; Surgical sutures; Synthase I; Synthesis Chemistry; Time; Tissue Engineering; Weight maintenance regimen; Work; X-Ray Crystallography; analog; base; biodegradable polymer; biomaterial compatibility; commercialization; cost; enzyme mechanism; enzyme substrate analog; in vivo; insight; metabolic engineering; mutant; phasin; phosphocellulose; protein protein interaction; public health relevance; unnatural amino acids

 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.

 描述（由申请人提供） 聚羟基脂肪酸酯 (PHA) 是由多种细菌在除碳之外的营养有限的生长条件下产生的多氧酯。由于其优异的生物相容性、生物降解性和多功能性，PHAs 已被开发用于医疗器械、药物输送和组织工程中的各种生物医学应用。 FDA 于 2009 年批准了 PHA 的首次医疗用途，即商品名为 TephaFLEX 的可吸收缝合线。然而，PHA生产成本较高，阻碍了其进一步发展和下游商业化。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特性的 PHA。为了促进这一点，本提案将重点关注 PHA 合酶 (PhaC) 和菜素蛋白 (PhaP)，它们对于 PHA 生产和所生产材料的特性至关重要。具体目标是：（1）表征PhaC在PHA生产和分子量（MW）控制中的机制。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比 III 类酶更具挑战性的 I 类合酶的链延长。还将努力寻找“其他因素”，建议参与使用转基因生物控制 PHA MW。蛋白质-蛋白质相互作用将通过下拉测定来鉴定强相互作用，并通过掺入光活性非天然氨基酸来鉴定弱相互作用。 PhaC 本身的 MW 控制也将通过合成类似物在体外进行研究，或通过识别链终止/重新启动过程中涉及的残基进行体内研究； (2)通过X射线晶体学获得PHA合酶的结构信息。与同一校区的一位出色的晶体学家 Geisbrecht 博士合作，来自不同细菌来源的合酶将被纯化，并在配体不存在和存在的情况下筛选结晶。我们与不可水解 CoA 类似物共结晶的初步结果为初始 PhaC 结构提供了一条清晰的途径。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宝贵见解，并实现我们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 的关系将使用各种结合测定以及补充有 PHA 生物合成途径的大肠杆菌在体外和体内进行表征。将首次通过荧光显微镜和点击化学的结合来监测体内颗粒的形成。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完善我们对 PHA 生产的理解非常重要。最终，这将使具有特定特性的 PHA 能够经济地生产用于医疗应用。我们的结果还将揭示不依赖模板的聚合的广泛反应，其机制仍然是个谜。