DISCOVERY AND CHARACTERIZATION OF PHOSPHONIC ACID BIOSYNTHESIS PATHWAYS

膦酸生物合成途径的发现和表征

• 批准号: 7249591
• 负责人: WILLIAM W METCALF
• 金额: $ 36.77万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7249591
• 关键词: Actinobacteria class; Actinomyces; Address; Alanine; Amino Acids; Anabolism; Area; Back; Binding; Biochemical; Biogenesis; Biological Assay; Biological Factors; Carbon; Categories; Class; Complement; Complex Mixtures; Computer software; Data Set; Defect; Detection; Development; Engineering; Enzymatic Biochemistry; Enzymes; Fourier Transform; Gene Cluster; Genes; Herbicides; Human; Hybrids; Illinois; Ions; Kinetics; Laboratories; Libraries; Ligase; Link; Mass Spectrum Analysis; Metabolic; Methylation; Molecular; Mutagenesis; Mutase; Object Attachment; Pathway interactions; Peptide Synthesis; Performance; Pharmaceutical Preparations; Phosphonic Acids; Phosphorus; Production; Property; Refractory; Research; Research Personnel; Role; Screening procedure; Series; System; Techniques; Time; Universities; Upper arm; analog; base; desire; experience; ginsenoside M1; inorganic phosphate; instrumentation; mass spectrometer; metabolomics; mutant; novel; peptide synthase; phosphinothricin; phosphonate; programs; scaffold; small molecule; thioester; tool

Compounds with C-P bonds represent an understudied class of natural products with proven and attractive properties as drugs for humans or herbicides. With stable carbon-phosphorus bonds, phosphonates and phosphinates are useful scaffolds that mimic phosphates and display desirable pharmacological properties. This major class of bioactive compounds will be addressed by an intensely collaborative team at the University of Illinois. The Kelleher Laboratory will leverage its core expertise in ultra-high performance Fourier-Transform Mass Spectrometry (FTMS) to implement instrumentation and tailored software for phosphonate compounds and biosynthetic intermediates in both targeted and discovery modes. For known phosphonates, both small molecule MS (metabolomics) and large molecule MS (FTMS-based assays), will be used to interrogate phosphonate intermediates both free and bound as thioesters to non-ribosomal peptide synthetases. Further, the compound K-26 will be linked to its biosynthetic gene cluster using a general fosmid-screening approach employing a highly automated FTMS-based screen. The enzymology underlying the biosynthesis of phosphinothricin tripeptide (PTT) will be elucidated. With emphasis on the PTT system, the Kelleher Laboratory will use its extensive experience in non-ribosomal peptide synthesis to dissect the timing of P- methylation and the role of a curious tandem thiolation domain in the biosynthetic assembly line. Armed with mechanistic understanding of the thiotemplate portion of this gene cluster, a series of phs mutants will be screened using large molecule FTMS to engineer the NRPS portion of the PTT cluster from S. vidriochromogenes, with production of unnatural PTT analogues in a heterologous producer, S. lividans, to follow. For both targeted analysis and discovery, the negative mass defect of phosphorus along with selective MS/MS detection approaches will require tailored software for "phosphonate-directed" metabolomics to filter large datasets emanating from ion trap/Fourier-Transform hybrid mass spectrometers operating at 7, 12, and 14.5Tesla. A particular strength of a MS approach to phosphonate discovery is that new compounds are identified based on structural features and not modes of biosynthesis or spectrum of activity in a bioassay. The FTMS approaches to engineered production and discovery of new phosphonates complements the many other strategies presented in this integratedP01.

具有C-P键的化合物代表了一类研究不足的天然产物， 作为人类药物或除草剂的特性。具有稳定的碳-磷键，膦酸酯和 次膦酸盐是模拟磷酸盐并显示所需药理学性质的有用支架。 这类主要的生物活性化合物将由一个密切合作的团队在 伊利诺伊大学。 凯莱赫实验室将利用其在超高性能傅里叶变换质量方面的核心专业知识， 光谱分析（FTMS），以实现仪器和定制的软件，为膦酸酯化合物和 生物合成中间体的目标和发现模式。对于已知的膦酸盐， 分子MS（代谢组学）和大分子MS（基于FTMS的测定）将用于询问 膦酸酯中间体既可以是游离的，也可以作为硫酯与非核糖体肽合成酶结合。 此外，化合物K-26将使用一般的fosmid-screening与其生物合成基因簇相关联。 采用高度自动化的基于FTMS的屏幕的方法。生物合成的酶学基础 膦丝菌素三肽（PTT）的活性。随着PTT系统的重点，凯莱赫 实验室将利用其在非核糖体肽合成方面的丰富经验来剖析P- 甲基化和一个奇怪的串联巯基化结构域在生物合成装配线中的作用。手持 通过对该基因簇的硫代模板部分的机械理解，将获得一系列phs突变体。 使用大分子FTMS筛选以工程化来自S. vidriochromogenes，与生产非天然PTT类似物在异源生产者，S。利维丹 跟随. 对于有针对性的分析和发现，磷的负质量缺陷沿着有选择性的 MS/MS检测方法将需要针对“磷酸盐导向”代谢组学的定制软件来过滤 从在7，12， 14.5特斯拉MS方法发现膦酸盐的一个特别优势是， 是基于结构特征而不是生物合成模式或活性谱来鉴定的。 生物测定工程化生产和发现新膦酸酯的FTMS方法 补充了本集成P01中提出的许多其他策略。