Engineering Photosystem I for Light-Driven Biocatalysis

用于光驱动生物催化的工程光系统 I

• 批准号: 2771571
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2771571
• 关键词: Engineering; Photosystem; Light; Driven; Biocatalysis

Reductive catalysis is a central element in a multitude of industrial and pharmaceutical processes, including 'functionalisation of bioactive compounds'1 and the reduction of alkenes2, but chemical catalysis is often unstable or functionally inert under standard conditions. This leads to a huge expenditure of energy and resources in maintaining the correct temperature and pressure environments for these reactions to be fruitful. In response to these limiters, there has been a great deal of successful research into alternative methods of catalysis, particularly in the fields of biocatalysis2 and photocatalysis3. Biocatalysis refers to the use of novel enzymes to catalyse reactions, while photocatalysis is a process in which photons are used to directly excite an inorganic semiconductor species, allowing it to catalyse the desired reaction4. Both approaches are currently used in industrial and pharmaceutical synthesis, however, greater reductive power could be achieved by merging these concepts. Photosystem I (PSI) is a biological molecular assembly for the ultimate photosynthetic reduction of NADP+ to NADPH, a reducing cofactor that powers metabolism in living systems. In recent years, there has been interest in engineering PSI/biocatalytic enzyme fusion proteins to harness the photocatalytic capabilities of PSI for use in novel reductive bio-photocatalysis5. Notably, PSI has been effectively fused with hydrogenase enzymes, resulting in highly reductive protein fusions capable of catalysing the reduction of H+ to elemental hydrogen using photons as the electron source6. Objectives To demonstrate that PSI can be used as a platform to develop novel light-driven enzymes that could be of biotechnological interest. To bioengineer a range of Photosystem I/reductive enzyme (PSI/RE) chimeric enzymes. To apply and develop directed evolution approaches to enhance the docking, substrate specificity, energetics, and/or kinetics of the PSI/RE fusions. Research A range of reductive enzymes will be fused with the PsaC subunit of a PSI from Synechocystis sp. PCC68036 to produce PSI/RE chimeras and undergo focused directed evolution. The reductive enzymes of interest will include industrial staples like members of the cytochrome p450 superfamily1, the ene-reductase group7, and a reductive dehalogenase8, as well as the carbon-fixing enzyme Crotonyl-CoA Carboxylase/Reductase (CCR)9 and a range of aldehyde/alcohol dehydrogenases of industrial interest10. In silico predictions of the pre- and post-fusion enzyme structures will be used to inform the best avenues for in vitro protein engineering, as well as suggest target regions for directed evolution. Diverse libraries of the PsaC/RE fusions will be produced using in vitro and in vivo mutagenesis techniques; error-prone PCR, site-saturation mutagenesis, and an EvolvR-derived11 approach developed in our lab. To this end, enzyme-specific approaches for screening and selection of light-driven chimera functionality will be developed, exploiting absorption, fluorescent probes, and colorimetry. Specifically, screening approaches will include Schiff's reagent12; substrate, product, or cofactor-specific spectrophotometry13,14; or photoactive PSI detection15. Ethical Considerations This research will necessitate genetic modification of Synechocystis. This could constitute an ethical problem should the modified cyanobacteria be released into the environment; however, the safety and operating procedures of our lab are compliant for work with genetically modified bacteria.

还原催化是许多工业和制药过程中的核心要素，包括“生物活性化合物的功能化”1和烯烃的还原2，但化学催化在标准条件下通常不稳定或功能惰性。这导致在维持正确的温度和压力环境以使这些反应富有成效方面消耗巨大的能量和资源。为了应对这些限制因素，人们对催化的替代方法进行了大量成功的研究，特别是在生物催化2和光催化3领域。生物催化是指使用新的酶来催化反应，而生物催化是一个过程，其中光子被用来直接激发无机半导体物种，使其能够催化所需的反应4。这两种方法目前都用于工业和药物合成，然而，通过合并这些概念可以实现更大的还原能力。光系统I（PSI）是用于最终将NADP+光合还原为NADPH的生物分子组装体，NADPH是为生命系统中的代谢提供动力的还原辅因子。近年来，人们对工程化PSI/生物催化酶融合蛋白以利用PSI的光催化能力用于新型还原性生物光催化产生了兴趣。值得注意的是，PSI已经有效地与氢化酶融合，导致高度还原的蛋白融合物能够使用光子作为电子源催化H+还原为元素氢6。目的证明PSI可以作为一个平台，开发新的光驱动酶，可能是生物技术的兴趣。利用生物工程技术合成一系列光系统I/还原酶（PSI/RE）嵌合酶。应用和开发定向进化方法，以增强PSI/RE融合的对接，底物特异性，能量学和/或动力学。研究一系列还原酶将与来自集胞藻PCC 68036的PSI的PsaC亚基融合以产生PSI/RE嵌合体并进行集中定向进化。感兴趣的还原酶将包括工业斯台普斯，如细胞色素p450超家族1、烯还原酶组7和还原性脱卤酶8的成员，以及碳固定酶巴豆酰辅酶A羧化酶/还原酶（CCR）9和一系列工业感兴趣的醛/醇脱氢酶10。融合前和融合后酶结构的计算机预测将用于为体外蛋白质工程提供最佳途径，并建议定向进化的靶区域。将使用体外和体内诱变技术、易错PCR、位点饱和诱变和我们实验室开发的EvolvR衍生方法11来产生PsaC/RE融合体的不同文库。为此，将开发用于筛选和选择光驱动嵌合体功能的酶特异性方法，利用吸收、荧光探针和比色法。具体而言，筛选方法将包括希夫试剂12;底物、产物或辅因子特异性荧光光谱法13，14;或光敏PSI检测15。伦理考虑这项研究将需要集胞藻的遗传修饰。这可能会构成一个道德问题，如果修改后的蓝藻被释放到环境中;然而，我们实验室的安全和操作程序是符合转基因细菌的工作。