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首页> 外文学位 >Transcriptomic and Physiological Analysis of a Recombinant Pyrococcus furiosus Strain Metabolically Engineered to Produce 3-Hydroxypropionate from CO2 and Maltose.
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Transcriptomic and Physiological Analysis of a Recombinant Pyrococcus furiosus Strain Metabolically Engineered to Produce 3-Hydroxypropionate from CO2 and Maltose.

机译：代谢工程改造以从二氧化碳和麦芽糖生产3-羟基丙酸酯的重组热球菌菌株的转录组学和生理分析。

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摘要

As a new paradigm for renewable fuels, Electrofuels expand the boundaries of traditional biofuels that rely on photosynthetic carbon fixation. Electrofuels could overcome the drawback inherent to biofuels approaches via direct utilization of CO2 to bypass energy intermediate sugar, more efficient non-photosynthetic CO2 fixation pathways and durable low potential energy sources. Here, we aim to metabolically engineer a hyperthermophilic archeaon, P. furiosus strain COM1, as a host for production of desired liquid fuels or valuable chemicals (e.g., 3-hydroxypropionate) by incorporation of the 3HP/4HB cycle from the thermoacidophile M. sedula. Sub-pathway 1 (SP1) of the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is composed of the heteromultimeric acetyl-CoA/propionyl-CoA carboxylase (E1) (encoded by Msed_0147, Msed_0148, and Msed_1375), malonyl-CoA reductase (E2) (encoded by Msed_0709), and malonic semialdehyde reductase (E3) (encoded by Msed_1993). These three enzymes sequentially convert HCO 3- and acetyl-CoA into 3-hydroxypropionate (3-HP). To achieve our goal, the in vitro study of SP1 was first performed to confirm all enzyme functions individually, and that if assembled could convert acetyl-CoA to 3-HP. The genes encoding these enzymes were produced by heterologous expression in E. coli or P. furiosus . In this process, the rate-limiting carboxylase in carbon fixation and its accessory enzyme biotin protein ligase (BPL) captured our specific attention, as did carbonic anhydrase (CA), the enzyme catalyzing conversion of CO2 to bicarbonate as substrate for carboxylase. Therefore, Msed_0390, and Msed_2010 were identified in the M. sedula genome to encode a functional CA and BPL. The in vitro work provided the basis for incorporating CA and BPL by metabolic engineering into recombinant P. furiosus to facilitate 3-HP/4-HB cycle function for production of fuels and chemicals. All three enzymes (E1, E2 and E3) were produced in recombinant form, with E2 and E3 in E. coli, and the three subunits encoding E1 in P. furiosus. Recombinant forms of M. sedula BPL and CA were also produced in E. coli. Formation of 3-HP was demonstrated in vitro by the assembly of recombinant E1, E2 and E3, using either acetyl-CoA or malonyl-CoA as substrate. The five M. sedula enzymes mentioned above were then metabolically engineered into P. furiosus strain COM1 to enable production of 3-hydroxypropionate (3-HP) from maltose and CO 2. P. furiosus was grown in a 2-liter fermentor at 95°C until late exponential phase, at which time the temperature was reduced to 72°C to initiate 3-HP production. The addition of genes encoding M. sedula CA and biotin BPL, led to two-fold higher 3-HP titers than in their absence. Furthermore, CO2 mass transfer was found to be rate-limiting, as 10-fold higher 3-HP concentrations (from 0.3 to 3 mM) were obtained when sparging and agitation rates were increased from 15 mL/min to 50 mL/min, and 250 rpm to 400 rpm, respectively. Transcriptomic analysis using a hybrid oligonucleotide microarray revealed that the M. sedula genes were among the most highly transcribed in the recombinant P. furiosus genome, both at 2.5 h and 40 h after the temperature shift. Monovalent cation /H+ antiporter subunits were found up-regulated in MW76, presumably involved in the export of 3-HP to maintain cytosolic homeostasis to prevent intracellular acidification. From 2.5 h to 40 h, the P. furiosus COM1 transcriptome showed significant down-regulation of genes involved in energy metabolism, amino acid biosynthesis, DNA metabolism and protein synthesis, while MW76 maintained relatively high cellular activity. Up-regulation of biotin synthesis genes in MW76 was also noted at 40h, presumably to provide sufficient biotin for biotinylation of the M. sedula carboxylase subunit. The results of this study will be critical in designing further genetic refinements to the MW76 strain and to coordinate these changes to bioprocessing strategies.
机译：作为可再生燃料的新范例，电子燃料扩展了依靠光合碳固定的传统生物燃料的范围。电燃料可通过直接利用CO2绕过能量中间糖，更有效的非光合CO2固定途径和持久的低电势能源来克服生物燃料方法固有的缺点。在这里，我们的目标是通过结合来自嗜热嗜酸小球藻的3HP / 4HB循环，对高嗜热古生菌P. furiosus菌株COM1进行代谢改造，以作为宿主生产所需的液体燃料或有价值的化学物质（例如3-羟基丙酸酯）。 。 3-羟基丙酸酯/ 4-羟基丁酸酯（3HP / 4HB）循环的子途径1（SP1）由异多聚乙酰-CoA /丙酰-CoA羧化酶（E1）（由Msed_0147，Msed_0148和Msed_1375编码）组成-CoA还原酶（E2）（由Msed_0709编码）和丙二醛半醛还原酶（E3）（由Msed_1993编码）。这三种酶依次将HCO 3-和乙酰辅酶A转化为3-羟基丙酸酯（3-HP）。为了实现我们的目标，首先进行了SP1的体外研究，以单独确认所有酶的功能，并且如果组装可以将乙酰辅酶A转化为3-HP。编码这些酶的基因是在大肠杆菌或激烈疟原虫中异源表达的。在此过程中，碳固定中的限速羧化酶及其辅助酶生物素蛋白连接酶（BPL）和碳酸酐酶（CA）引起了我们的特别关注，碳酸酐酶（CA）催化将CO2转化为碳酸氢根作为羧化酶的底物。因此，在M. sedula基因组中鉴定出Msed_0390和Msed_2010，以编码功能性CA和BPL。体外工作提供了通过代谢工程将CA和BPL整合到重组P. furiosus中的基础，以促进3-HP / 4-HB循环功能来生产燃料和化学品。所有三种酶（E1，E2和E3）均以重组形式产生，其中E2和E3在大肠杆菌中产生，而三个亚基在Furiosus中编码E1。景天支原体BPL和CA的重组形式也在大肠杆菌中产生。通过使用乙酰辅酶A或丙二酰辅酶A作为底物组装重组E1，E2和E3体外证明了3-HP的形成。然后，将上述五种小枝麻风杆菌酶经代谢工程改造为激烈的毕赤酵母菌株COM1，以使麦芽糖和CO 2产生3-羟基丙酸酯（3-HP）。在2升的发酵罐中于95°C下生长剧烈的毕赤酵母。 C直到指数末期，此时温度降低到72°C以启动3-HP生产。编码景天分枝杆菌CA和生物素BPL的基因导致3-HP滴度比不存在时高两倍。此外，发现二氧化碳的传质受到速率的限制，因为在将喷射和搅拌速率从15 mL / min增加到50 mL / min时，3-HP浓度提高了10倍（从0.3到3 mM），并且250 rpm至400 rpm。使用杂交寡核苷酸微阵列的转录组学分析显示，在温度变化后的2.5 h和40 h时，景天支原体基因在重组重组毕赤酵母基因组中转录度最高。在MW76中发现单价阳离子/ H +反转运蛋白亚基上调，可能参与了3-HP的输出，以维持细胞内稳态，防止细胞内酸化。从2.5 h至40 h，狂犬病菌COM1转录组显示出与能量代谢，氨基酸生物合成，DNA代谢和蛋白质合成有关的基因的显着下调，而MW76保持相对较高的细胞活性。还在第40小时注意到了MW76中生物素合成基因的上调，大概是为景天支气管羧化酶亚基的生物素化提供了足够的生物素。这项研究的结果对于为MW76菌株设计进一步的遗传改良并协调这些变化与生物加工策略至关重要。

著录项

  • 作者

    Lian, Hong.;

  • 作者单位

    North Carolina State University.;

  • 授予单位 North Carolina State University.;
  • 学科 Engineering Chemical.;Biology Genetics.;Biology Molecular.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 266 p.
  • 总页数 266
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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