Functional and Structural Analysis of Algal Hydrogenase Combinatorial Mutants (TSE03-D)

藻类氢化酶组合突变体的功能和结构分析（TSE03-D）

• 批准号: 0328187
• 负责人: Dianne Ahmann
• 金额: $ 24万
• 依托单位: Colorado School of Mines
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0328187&HistoricalAwards=false
• 关键词: Functional; Structural; Analysis; Algal; Hydrogenase

Molecular hydrogen is a promising energy source due to its clean combustion and potential for sustainability. Among hydrogen producing technologies, photosynthetic mechanisms are particularly interesting because of their potentially low energy requirements, as exemplified by the Fe hydrogenase system in the green alga Chlamydomonas reinhardtii. However, hydrogen production in C. reinhardtii is inhibited both transcriptionally and post-translationally by molecular oxygen. These features pose a significant challenge to the development of commercial algal hydrogen generation, which is estimated to require a 70 to 720-fold improvement in oxygen tolerance of the hydrogenase enzyme itself. This project is intended to yield such an enzyme, using the approach of combinatorial mutagenesis (DNA shuffling) to generate mutants that cannot be produced by other methods. Additional objectives are to develop a novel colorimetric assay for hydrogen production into an automated, high-throughput screen to evaluate mutant libraries for optimal diversity before undertaking in-depth analyses and to investigate a promising new method known as Random Chimeragenesis on Transient Templates (RACHITT) that may generate even more useful libraries. Most-improved genes will be recovered and re-shuffled as necessary, and functional and structural modeling studies will be conducted on the best mutants to reveal mechanisms of improvement. The intended product is an oxygen-tolerant Fe hydrogenase that will ultimately facilitate commercial algal hydrogen production.

由于其清洁燃烧和可持续发展的潜力，分子氢是一种很有前途的能源。在制氢技术中，光合作用机制特别令人感兴趣，因为它们可能需要较低的能量，例如绿藻衣藻中的铁氢酶系统。然而，莱茵梭菌的产氢在转录和翻译后都受到分子氧的抑制。这些特点对商业藻类制氢的发展构成了重大挑战，据估计，这需要氢酶本身的耐氧性提高70至720倍。该项目旨在生产这样一种酶，使用组合突变(DNA改组)的方法来产生其他方法无法产生的突变。其他目标是开发一种新的产氢比色分析方法，用于自动化、高通量筛选，以在进行深入分析之前评估突变文库的最佳多样性，并研究一种名为瞬时模板随机嵌合发生(RACHITT)的有前景的新方法，该方法可能会产生更有用的文库。改进最多的基因将被恢复并在必要时重新洗牌，并将对最好的突变体进行功能和结构建模研究，以揭示改进的机制。预期的产品是一种耐氧铁氢酶，最终将促进藻类氢气的商业生产。