Design of a light-driven biocatalyst for biofuel production

光驱动的反应在生物化学中很少见，到目前为止只有三种光依赖的酶被表征。然而，可见光加速化学反应提供了环境友好的化学合成途径，这可能是实用的，并可在工业生产中使用。一种利用蓝光将脂肪酸转化为碳氢化合物的藻类光酶的发现，表明了迄今为止通过辅因子依赖的酶进行生物技术有用的光化学的隐藏空间。事实上，其他人已经证明，在适当的条件下，一系列依赖辅因子的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是什么，并将利用天然脱羧酶FDC(广泛存在的UbiD家族的成员)。后一种酶在体内利用了一种前烯基化的辅因子(PrFMN)，但在体外可以与未修饰的黄素(FMN)结合。重要的是，底物仍然能够结合到黄素辅因子附近，但在黑暗条件下没有观察到反应。因此，存在两个关键因素：底物/黄素的紧密并列和一个朝向脱羧基的活性部位。令人鼓舞的是，在光照下，我们发现一系列产品是以依赖于光的方式形成的。因此，我们理想地准备开发非自然的和强大的光依赖脱羧酶使用prFMN依赖的UbiD酶家族作为合适的模板。我们的初步研究将集中在一系列条件下的产品鉴定和定量(在厌氧/好氧条件下变化的光波长和强度等)。我们的初步数据已经表明，氧的存在会改变产品的轮廓，这表明在氧的存在下，会发生不同的氧化光化学。然后，我们将通过探索一系列影响活性部位组成和黄素结合环境的FDC变异酶来继续我们的研究。在这种情况下，所使用的酶是一个理想的测试对象，因为原子分辨率的晶体结构通常是在我们的实验室获得的。因此，我们能够通过结合结晶学和DFT计算来研究机械细节中感兴趣的变体，就像我们以前对自然的prFMN依赖反应所做的那样。活性中心的性质(根据芳香族侧链或酸碱残基的存在)将以合理的方式改变，以探索其对光依赖催化的影响。我们的长期目标是产生一种高效的FMN和光相关的UBD，它可以很容易地应用于各种衬底，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，得到的工业支持进一步证明了这一点。

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.