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

分子氢由于其清洁燃烧和可持续发展的潜力，是一种很有前途的能源。在制氢技术中，光合作用机制特别有趣，因为它们潜在的低能量需求，绿藻衣藻（Chlamydomonas reinhardtii）中的铁氢化酶系统就是一个例子。然而，C. reinhardtii的产氢在转录和翻译后都受到分子氧的抑制。这些特征对商业化藻类制氢的发展提出了重大挑战，据估计，这需要将氢化酶本身的氧耐受性提高70至720倍。本项目旨在生产这样一种酶，使用组合诱变（DNA洗牌）的方法来产生其他方法无法产生的突变体。其他目标是开发一种新的比色法，用于自动化的高通量筛选，以在进行深入分析之前评估突变文库的最佳多样性，并研究一种有前途的新方法，称为瞬时模板随机嵌合（RACHITT），该方法可能产生更有用的文库。大多数改良基因将根据需要进行恢复和重组，并对最佳突变体进行功能和结构建模研究，以揭示改良机制。预期的产品是一种耐氧铁氢化酶，最终将促进商业藻类制氢。

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.