组装和工程BioBricks开发一个高效的NADH再生系统的工程
Feng Cheng1,2, Cheng-Jiao Wang1,2, Xiao-Xiao Gong1,2
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China.
Applied and environmental microbiology
|December 11, 2024
概括
这项研究使用酒精脱酶 (ADH) 和BioBricks组件设计了一种高效的NADH再生系统. 优化的系统显著提高了生物催化剂的催化效率和基因表达,在拉式除草剂合成中实现了超过95%的产量.
科学领域:
- 生物催化和合成生物学
- 酶工程和辅助因子再生
背景情况:
- 辅因子再生对于有机合成和制药中的氧化还原生物催化剂至关重要.
- 基于酒精脱酶 (ADH) 的系统在现场提供NAD(P) H再生,但面临着在大肠杆菌中低活性和表达的挑战.
- 以前的努力集中在ADH发现和蛋白质工程上,忽视了其他关键的系统组件.
研究的目的:
- 通过BioBricks的组装和工程开发一个高效的NADH再生系统.
- 通过使用半理性设计来提高Geobacillus stearothermophilus ADH (GstADH) 的催化效率.
- 优化核糖体结合部位 (RBS) 序列,以增加ADH基因表达.
主要方法:
- 生物组件 (促进器,RBS,基因,终止器) 用于构建NADH再生系统.
- 半理性设计GstADH以提高其催化效率.
- RBS库的选和优化,以提高ADH的翻译速度.
- 工程系统的应用对l-phosphinothricin的不对称生物合成.
主要成果:
- 产生了GstADH变体,其催化效率增加了2.1倍.
- 一个优化的RBS导致ADH基因转化率增加了3.2倍.
- 设计的NADH再生系统实现了超过2s-1的NADH生成速度,即使在较低的NADH+度 (0.1mM) 中也是如此.
- 与之前的系统相比,该系统的整体性能提高了6.7倍.
- 在l-phosphinothricin的不对称生物合成中成功应用,产量>95%.
结论:
- 开发的基于BioBricks的NADH再生系统非常高效和强大.
- 这种工程系统代表了生物催化剂中NAD(P) H再生的重大进步.
- 该系统对各种依赖NADH的生物催化过程具有前景,特别是在精细化学合成中.
相关概念视频
Role of Reduced Coenzymes NADH and FADH₂
11.2K
The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers that will be considered originate from the B vitamin group and are derivatives of nucleotides; they are...
11.2K
Electron Transport Chain: Complex I and II
11.5K
The mitochondrial electron transport chain (ETC) is the main energy generation system in the eukaryotic cells. However, mitochondria also produce cytotoxic reactive oxygen species (ROS) due to the large electron flow during oxidative phosphorylation. While Complex I is one of the primary sources of superoxide radicals, ROS production by Complex II is uncommon and may only be observed in cancer cells with mutated complexes.
ROS generation is regulated and maintained at moderate levels necessary...
ROS generation is regulated and maintained at moderate levels necessary...
11.5K
The Z-Scheme of Electron Transport in Photosynthesis
9.9K
The light reactions of photosynthesis assume a linear flow of electrons from water to NADP+. During this process, light energy drives the splitting of water molecules to produce oxygen. However, oxidation of water molecules is a thermodynamically unfavorable reaction and requires a strong oxidizing agent. This is accomplished by the first product of light reactions: oxidized P680 (or P680+), the most powerful oxidizing agent known in biology. The oxidized P680 that acquires an electron from the...
9.9K
The Supercomplexes in the Crista Membrane
2.5K
The mitochondrial cristae membrane is the primary site for the oxidative phosphorylation (OXPHOS) process of energy conversion mediated through respiratory complexes I to V. These complexes have been widely studied for decades, and it has been proven that they form supramolecular structures called respiratory supercomplexes (SC). These higher-order complexes may be crucial in maintaining the biochemical structure and improving the physiological activity of the individual complexes while...
2.5K
The Electron Transport Chain
16.1K
The electron transport chain or oxidative phosphorylation is an exothermic process in which free energy released during electron transfer reactions is coupled to ATP synthesis. This process is a significant source of energy in aerobic cells, and therefore inhibitors of the electron transport chain can be detrimental to the cell's metabolic processes.
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q...
Inhibitors of the electron transport chain
Rotenone, a widely used pesticide, prevents electron transfer from Fe-S cluster to ubiquinone or Q...
16.1K
Electron Transport Chain: Complex III and IV
7.0K
During the electron transport chain, electrons from NADH and FADH2 are first transferred to complexes I and II, respectively. These two complexes then transfer the electrons to ubiquinol, which carries them further to complex III. Complex III passes the electrons across the intermembrane space to Cyt c, which carries them further to complex IV. Complex IV donates electrons to oxygen and reduces it to water. As electrons pass through complexes I, III, and IV, the energy released aids the pumping...
7.0K


