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首页> 外文学位 >Efficient experimental design and micro-scale medium enhancement of 6-deoxyerythronolide B production through Escherichia coli.
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Efficient experimental design and micro-scale medium enhancement of 6-deoxyerythronolide B production through Escherichia coli.

机译：通过大肠杆菌生产6-脱氧赤藓醇B的高效实验设计和微型培养基增强。

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摘要

As of 2007, 63% of small molecule drugs developed to battle cancer and 54% of drugs against infectious diseases were of natural origin (Newman and Cragg 2007). The recent use of heterologous hosts to produce natural products has shown significant potential, although limitations still exist regarding optimal production titers. In this study, we utilize micro-scale cultures and well-defined screening methods to identify key medium components that influence the heterologous production of the complex polyketide 6-deoxyerythronolide B (6dEB), the parent macrolactone precursor of erythromycin, through E. coli.; As a preliminary step, a thorough evaluation of a mass spectrometer (MS), an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD) were used to analyze erythromycin and 6-dEB. The work highlights the capabilities of each detector to analyze polyketide compounds that do not possess a natural chromophore. Each detector was evaluated based upon limit of detection (LOD), dynamic range, and precision in the context of polyketide analysis. Due to its low LOD, wide dynamic range, and ability to provide molecular weight information, the MS was deemed the best detection option for analysis of low-concentration, poorly identified polyketide compounds. Alternatively, both the CAD and ELSD systems studied showed better precision and accuracy. The ELSD demonstrated the best precision at 3%, but its LOD was limited to concentrations primarily greater than or equal to 1 mg/L-1. The Corona CAD demonstrated a LOD (0.012 mg/L-1) and dynamic range comparable to mass spectroscopy and, therefore, serves as a more cost efficient alternative for polyketide production schemes with low titers.; Using an ELSD with an online HPLC separation, an 8-run Plackett-Burman influenced screening method was conducted using micro-scale cultures. It was determined that tryptone had a significant effect on 6dEB production and could supplement substrate requirements and improve recombinant protein levels, including the essential deoxyerythronolide B synthase proteins which catalyze 6dEB conversion. As a result, we demonstrate the use of micro-scale experiments to effectively model larger-scale cultures and describe an enhanced medium which generated over 160 mg/L-1 6dEB (a 22-fold improvement over current culture media). Additional feeding experiments provided new insight and understanding related to the heterologous production of 6dEB from E. coli. These results will allow further studies to explore alternate pathways to 6dEB production. Long term goals are to sufficiently produce therapeutic natural products using these newly developed techniques.
机译：截至2007年，已开发出63％的抗癌小分子药物和54％的抗传染病药物是天然来源的（Newman and Cragg 2007）。异源宿主最近用于生产天然产物的用途已显示出巨大的潜力，尽管在最佳生产效价方面仍然存在局限性。在这项研究中，我们利用微观规模的文化和明确的筛选方法来确定关键的培养基成分，这些成分会通过大肠杆菌影响异源聚酮6-脱氧赤藓醇B（6dEB）（红霉素的母体大内酯前体）的异源生产。 ;作为第一步，对质谱仪（MS），蒸发光散射检测器（ELSD）和带电气溶胶检测器（CAD）进行了全面评估，以分析红霉素和6-dEB。这项工作强调了每个检测器分析不具有天然发色团的聚酮化合物的能力。在聚酮化合物分析的背景下，根据检测限（LOD），动态范围和精度评估每个检测器。由于其低检测限，宽动态范围和提供分子量信息的能力，因此质谱被认为是分析低浓度，鉴定较差的聚酮化合物的最佳检测选择。另外，研究的CAD和ELSD系统都显示出更好的精度和准确性。 ELSD的最佳精度为3％，但其LOD限于主要大于或等于1 mg / L-1的浓度。电晕CAD的LOD（0.012 mg / L-1）和动态范围可与质谱媲美，因此可作为滴定度低的聚酮化合物生产方案的更具成本效益的替代方案。使用具有在线HPLC分离功能的ELSD，使用微型培养物进行了8次运行的Plackett-Burman影响的筛选方法。已确定胰蛋白on对6dEB的产生具有显著作用，并且可以补充底物需求并提高重组蛋白水平，包括催化6dEB转化的必需脱氧赤藓醇B合成酶蛋白。结果，我们证明了使用微型实验对大型培养物进行有效建模，并描述了产生超过160 mg / L-1 6dEB的增强型培养基（比当前培养基提高了22倍）。额外的喂养实验提供了与大肠杆菌异源产生6dEB有关的新见识和理解。这些结果将使进一步的研究探索产生6dEB的替代途径。长期目标是使用这些新开发的技术来充分生产天然治疗性产品。

著录项

  • 作者

    Pistorino, Michael.;

  • 作者单位

    Tufts University.$bChemical and Biological Engineering.;

  • 授予单位 Tufts University.$bChemical and Biological Engineering.;
  • 学科 Engineering Chemical.
  • 学位 M.S.
  • 年度 2008
  • 页码 65 p.
  • 总页数 65
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化工过程（物理过程及物理化学过程）;
  • 关键词

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