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首页> 外文学位 >Differentiation engineering of Clostridium acetobutylicum for enhancing bioprocess characteristics.
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Differentiation engineering of Clostridium acetobutylicum for enhancing bioprocess characteristics.

机译：丙酮丁醇梭菌的分化工程以增强生物过程特性。

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摘要

Clostridia species can potentially be industrial-hosts for sustainable biofuel and chemical production. However, the metabolic engineering tools for clostridia are limiting. Subsequently, we developed and applied differentiation engineering to improve butanol production in Clostridium acetobutylicum.;Previous analysis of the spo0A knockout, the master transcriptional regulator (Harris et al., 2002), and detailed transcriptional analysis of clostridial sporulation (Jones et al., 2008), suggest that a transcription regulator, downstream (temporally) of Spo0A, likely blocks sporulation and preserves solventogenesis. Subsequently, we knocked out sigE and sigG by a novel gene disruption approach. The DeltasigE strain stalled sporulation prior to stage II, resulting in only vegetative-like phenotypes. The DeltasigE cells did not synthesize granulose, but did produce solvents, albeit in a bifurcating mode (normal versus low levels) that was dependent on the physiological state of the inoculum. The Delta sigG strain stalled sporulation at stage V, exhibiting engulfment and partial cortex and spore-coat formation, and displayed enhanced solventogenesis.;As mentioned, I invented a novel gene disruption approach that is applicable to all Clostridia. Previous attempts to disrupt genes in any Clostridia were inefficient, which we argue is due to the lack of a resolvase protein. By over-expressing the Bacillus subtilis resolvase, RecU, from a replicating gene disruption plasmid, we successfully knocked out spo0A by Campbell-like double crossover integration and sigE and sigG by single crossover integration. Characteristics of the disrupting plasmid that affected the efficiency of integration were investigated. Integration was most efficient when the repL origin of replication was in the opposite coding strand of the disrupting antibiotic marker, and when the disrupting antibiotic marker was in the same coding strand as the gene targeted for disruption.;Lastly, we attempted to determine the minimal genetic loci on the pSOL1 megaplasmid necessary for sporulation by constructing a functional pSOL1 complementation library in degenerate strains of C. acetobutylicum. Assuming the loci could be determined, they could serve as additional differentiation engineering targets to abolish sporulation and maintain solventogenesis. Thus far, we have been unsuccessful, and I suggest a pSOL1 specific gene knockout library in wild-type cells to determine these loci.
机译：梭菌可能成为可持续生物燃料和化学生产的工业宿主。然而，用于梭菌的代谢工程工具是有限的。随后，我们开发并应用了分化工程技术来提高丙酮丁醇梭菌中丁醇的产量。先前对spo0A敲除，主转录调节子（Harris等，2002）的分析以及对梭菌孢子形成的详细转录分析（Jones等，2002）。 2008年）表明，Spo0A下游（暂时）的转录调节子可能阻断孢子形成并保留溶剂生成。随后，我们通过一种新颖的基因破坏方法敲除了sigE和sigG。 DeltasigE菌株在II期之前停止了孢子形成，仅导致了类似营养的表型。 DeltasigE细胞不能合成颗粒，但会产生溶剂，尽管其分叉模式（正常与低水平）取决于接种物的生理状态。 Delta sigG菌株在第五阶段停止了孢子形成，表现出吞噬，部分皮层和孢子被膜形成，并显示出增强的溶剂生成。;如上所述，我发明了一种适用于所有梭菌的新型基因破坏方法。先前破坏任何梭状芽胞杆菌基因的尝试均无效，我们认为这是由于缺乏分辨酶蛋白。通过从复制基因破坏质粒中过表达枯草芽孢杆菌分离酶RecU，我们成功地通过坎贝尔样双交换整合敲除了spo0A，并通过单交换整合敲除了sigE和sigG。研究了影响整合效率的破坏质粒的特征。当repL复制起点位于破坏性抗生素标记的相反编码链中，并且破坏性抗生素标记与破坏基因位于同一编码链时，整合是最有效的方法；最后，我们尝试确定最小的通过在退化的丙酮丁醇梭菌菌株中构建功能性pSOL1互补文库，在孢子形成所必需的pSOL1大质粒上找到一个基因座。假设可以确定基因座，它们可以作为其他分化工程目标来消除孢子形成并维持溶剂生成。到目前为止，我们一直没有成功，我建议在野生型细胞中使用pSOL1特异性基因敲除文库来确定这些基因座。

著录项

  • 作者

    Tracy, Bryan Patrick.;

  • 作者单位

    Northwestern University.;

  • 授予单位 Northwestern University.;
  • 学科 Biology Molecular.;Alternative Energy.;Biology Microbiology.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 261 p.
  • 总页数 261
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:30

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