Design of a light-driven biocatalyst for biofuel production

用于生物燃料生产的光驱动生物催化剂的设计

• 批准号: 2627015
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2627015
• 关键词: Design; light; driven; biocatalyst; biofuel

Light driven reactions are rare in biochemistry, and only three light-dependent enzymes have been characterised to date. However, acceleration of chemical reactions by visible light offers environmentally friendly routes to chemical synthesis that may be practical and scalable for use in industrial manufacture. The discovery of an algal photoenzyme that uses blue light to convert fatty acids into hydrocarbons suggest a hitherto hidden scope for biotechnologically useful photochemistry by means of cofactor-dependent enzymes. Indeed, others have shown that non-natural reactions can occur for a range of cofactor-dependent enzymes under appropriate conditions. We seek to investigate what are the minimal requirements for a light-driven decarboxylase and will make use of the natural decarboxylase enzyme Fdc (a member of the widespread UbiD family). The latter enzyme makes use of a prenylated cofactor (prFMN) in vivo, but can be made to bind unmodified flavin (FMN) in vitro. Crucially, substrates are still able to bind adjacent to the flavin cofactor but no reaction is observed under dark conditions. Hence, two key elements are present: close juxtaposition of substrate/flavin and an active site geared towards decarboxylation. Encouragingly, upon illumination we find a range of products is formed in a light-dependent manner. Hence, we are ideally poised to develop non-natural and robust light-dependent decarboxylases using the prFMN-dependent UbiD enzyme family as a suitable template. Our initial investigations will focus on product identification and quantification under a range of conditions (varying light wavelength and intensity, under anaerobic / aerobic conditions etc). Our initial data already suggest that the presence of oxygen alters the product profile, suggesting distinct oxidative photochemistry occurs in presence of oxygen. We will then continue our investigations by probing a range of variant Fdc enzymes that affect active site composition and the flavin binding environment. The enzyme used is an ideal test subject in this case, as crystal structures to atomic resolution are routinely obtained in our laboratory. Thus, we are able to investigate variants of interest in mechanistic detail by combining crystallography with DFT calculations as we have done previously for the natural prFMN dependent reaction. The nature of the active site (in terms of the presence of aromatic side chains or acid/base residues) will be altered in a rational way to explore the affect on light-dependent catalysis. The long term goal is to generate an efficient FMN and light dependent UbiD that can readily be applied to range of substraets to support industrial application. As such, the project is directly within the industrial biotechnology remit, further demonstrated by the industrial support this receives.

光驱动反应在生物化学中很少见，迄今为止仅对三种光依赖性酶进行了表征。然而，可见光加速化学反应提供了环保的化学合成途径，这对于工业制造来说可能是实用且可扩展的。使用蓝光将脂肪酸转化为碳氢化合物的藻类光酶的发现表明，通过辅因子依赖性酶，生物技术上有用的光化学具有迄今为止隐藏的范围。事实上，其他人已经表明，在适当的条件下，一系列辅因子依赖性酶可以发生非自然反应。我们试图研究光驱动脱羧酶的最低要求，并将利用天然脱羧酶 Fdc（广泛使用的 UbiD 家族的成员）。后一种酶在体内利用异戊二烯化辅因子 (prFMN)，但可以在体外结合未修饰的黄素 (FMN)。至关重要的是，底物仍然能够与黄素辅因子相邻结合，但在黑暗条件下没有观察到反应。因此，存在两个关键要素：底物/黄素的紧密并置和面向脱羧的活性位点。令人鼓舞的是，在光照下，我们发现一系列产品以依赖于光的方式形成。因此，我们完全准备好使用 prFMN 依赖性 UbiD 酶家族作为合适的模板来开发非天然且强大的光依赖性脱羧酶。我们的初步研究将集中在一系列条件下（不同的光波长和强度、厌氧/有氧条件等）下的产品鉴定和定量。我们的初步数据已经表明，氧气的存在会改变产品的特性，表明在氧气存在的情况下会发生明显的氧化光化学反应。然后，我们将通过探测一系列影响活性位点组成和黄素结合环境的变体 Fdc 酶来继续我们的研究。在这种情况下，所使用的酶是理想的测试对象，因为我们的实验室通常可以获得原子分辨率的晶体结构。因此，我们能够通过将晶体学与 DFT 计算相结合来研究机械细节中感兴趣的变体，就像我们之前对自然 prFMN 依赖反应所做的那样。活性位点的性质（就芳香族侧链或酸/碱残基的存在而言）将以合理的方式改变，以探索对光依赖性催化的影响。长期目标是生成高效的 FMN 和光相关 UbiD，可以轻松应用于一系列子系统以支持工业应用。因此，该项目直接属于工业生物技术的范围，这一点得到了工业支持的进一步证明。