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

PHA 生物合成的分子研究：医用可生物降解聚合物的生产

• 批准号: 9915947
• 负责人: Ping Li
• 金额: $ 28.56万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9915947
• 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; Industrialization; 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; in vivo monitoring; insight; metabolic engineering; mutant; phasin; phosphocellulose; protein protein interaction; public health relevance; synergism; 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） 多羟基烷酸酯（PHAS）是在营养限制的生长条件下通过多种细菌产生的多氧植物。由于其出色的生物相容性，生物降解性和多功能性，PHA已开发用于医疗设备，药物输送和组织工程中的各种生物医学应用。 FDA在2009年批准了PHAS在商品名称Tephaflex下的可吸收缝合线。但是，PHA生产的高成本一直是他们进一步发展和下游商业化的障碍。我们的目标是识别和理解完整的PHA生物合成机制，以便可以在经济上生产具有定义特性的PHA。为了促进这一点，目前的建议将集中在PHA合酶（PHAC）和Phasin蛋白（PHAP）上，这是PHA产生和产生材料的特性的关键。具体目的是：（1）表征PHAC在PHA产生和控制分子量（MW）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研究I类合成酶的链伸长，这比III类酶更具挑战性。还将努力寻找提议使用一般修改的生物来控制PHA MW的“其他因素”。蛋白质 - 蛋白质相互作用将通过下拉测定法进行较强的相互作用，并通过将光活性非天然氨基酸纳入弱相互作用。 PHAC本身的MW对照也将通过合成类似物或体内进行体外研究，通过识别链条终止/重新发射过程中涉及的残基； （2）通过X射线晶体学获得有关PHA合酶的结构信息。与在同一校园里有成就的晶体学者Geisbrecht博士合作，在不存在和存在配体的情况下，将纯化和筛选来自不同细菌来源的合成酶，以进行结晶。我们与不可水解的COA类似物共结晶的初步结果为初始PHAC结构提供了清晰的路径。这种X射线结构的可用性将为我们提供有关底物识别和酶机制的宝贵见解，并实现了我们的蛋白质工程的长期目标； （3）表征PHAP在PHA产生和颗粒形成中的作用。 PHAC和PHAP的关系将在体外和体内进行特征，并使用各种结合测定法，并与补充了PHA生物合成途径的大肠杆菌。通过荧光显微镜和点击化学的组合，将首次在体内监测颗粒的形成。阐明PHAC，PHAP，“其他因素”和颗粒（PHA）在分子水平上的作用和关系对于完成我们对PHA产生的理解非常重要。最终，这将允许具有定义特性的PHA用于医疗应用。我们的结果还将阐明与模板无关聚合的宽度反应，在该反应中，该机理仍然存在神秘性。