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In Planta Expression of Heterologous Cell Wall-Degrading Enzymes.

机译：在植物中异源细胞壁降解酶的表达。

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摘要

For several reasons, ethanol derived from cornstarch cannot meet the domestic demand for liquid transportation fuel. Cellulosic biomass is an untapped source of the sugars used to produce ethanol and more complex biofuels. Compared to starch, cellulosic biomass requires a higher loading and wider variety of enzymes for hydrolysis into simple sugars. This research explores in planta production of cellulosic biomass-degrading enzymes.;Genes for an endoglucanase and xylanase from the thermophilic bacteria Acidothermus cellulolyticus were codon optimized for expression in the tobacco relative Nicotiana benthamiana. Transient expression was induced by infiltrating N. benthamiana plants with Agrobacterium tumefaciens containing the gene(s) of interest. Three different expression systems were tested, including a 35S-driven system, a Cucumber Mosiac Virus replicase-based system (CMVva), and a Tobacco Mosaic Virus replicase-based system (TRBO). Co-expression of the gene-silencing suppressor p19 from Tomato Bushy Stunt Virus improved the expression of 35S-driven endoglucanase from 5.4 +/- 0.4 to 15.2 +/- 2.0 mg/kg fresh weight of plant tissue. Co-expression of p19 with the replicase-based systems was necessary for endoglucanase expression, resulting in 6.3 +/- 0.9 from CMVva and 7.0 +/- 0.9 from TRBO. For the xylanase gene, only the 35S-driven system was tested, and co-expression with p19 or an alternative gene silencing suppressor HcPro from Tobacco Etch Virus improved expression up to 6.9 +/- 1.5 mg/kg fresh weight of plant tissue.;The codon-optimized endoglucanase gene was expressed in the methylotrophic yeast Pichia pastoris and secreted into culture media at 550 mg/L culture supernatant. The Pichia and N. benthamiana-produced endoglucanases had the same optimal temperature and pH, as well as the same Michaelis-Menten constant (KM = 105 &mgr;M). The highest-yielding strain of Pichia is available to produce endoglucanase as a reference for future research.;A commercial scale process for in planta production of cellulosic biomass-degrading enzymes was modeled in SuperPro. It was determined that a transient expression process was not economically feasible. An economically feasible process using stably transformed plants expressing a battery of cellulosic biomass-degrading enzymes and incorporating up-to-date enzyme loading assumptions from industrial enzyme manufacturers was modeled and sensitivity analyses were conducted.
机译：由于多种原因，源自玉米淀粉的乙醇不能满足国内对液体运输燃料的需求。纤维素生物质是用于生产乙醇和更复杂的生物燃料的糖的未开发来源。与淀粉相比，纤维素生物质需要更高的负载量和更多种类的酶才能水解为单糖。这项研究探索了纤维素生物质降解酶在植物中的生产。对嗜热嗜酸菌解热嗜酸菌的内切葡聚糖酶和木聚糖酶基因进行了密码子优化，以在烟草亲本烟草中表达。通过用含有目的基因的根癌农杆菌渗入本氏烟草植物来诱导瞬时表达。测试了三种不同的表达系统，包括35S驱动系统，基于黄瓜花叶病毒复制酶的系统（CMVva）和基于烟草花叶病毒复制酶的系统（TRBO）。从番茄浓密特技病毒中共表达基因沉默抑制剂p19，可将35S驱动的内切葡聚糖酶的表达从5.4 +/- 0.4增至15.2 +/- 2.0 mg / kg植物组织鲜重。 p19与基于复制酶的系统的共表达对于内切葡聚糖酶表达是必需的，导致CMVva为6.3 +/- 0.9，TRBO为7.0 +/- 0.9。对于木聚糖酶基因，仅测试了35S驱动系统，与烟草蚀刻病毒的p19或其他基因沉默抑制剂HcPro共表达可将表达提高至植物组织鲜重达6.9 +/- 1.5 mg / kg。密码子优化的内切葡聚糖酶基因在甲基营养酵母巴斯德毕赤酵母中表达，并以550 mg / L的培养上清液分泌到培养基中。毕赤酵母和本氏烟草生产的内切葡聚糖酶具有相同的最佳温度和pH，以及相同的Michaelis-Menten常数（KM = 105μM）。毕赤酵母产量最高的菌株可用于生产内切葡聚糖酶，以备将来研究之用。在SuperPro中模拟了植物规模生产纤维素生物质降解酶的商业规模过程。确定瞬时表达过程在经济上不可行。使用稳定转化的植物表达一组纤维素生物质降解酶并结合工业酶生产商的最新酶负载假设，对经济可行的过程进行了建模，并进行了敏感性分析。

著录项

  • 作者

    Lindenmuth, Benjamin E.;

  • 作者单位

    University of California, Davis.;

  • 授予单位 University of California, Davis.;
  • 学科 Biology Cell.;Agriculture Plant Culture.;Chemistry Biochemistry.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 239 p.
  • 总页数 239
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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