Engineering Photosystem I for Light-Driven Biocatalysis

还原催化是众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。这就需要花费大量的能量和资源来维持正确的温度和压力环境，以使这些反应富有成效。为了应对这些限制因素，人们对替代催化方法进行了大量成功的研究，特别是在生物催化和光催化领域。生物催化是指使用新的酶来催化反应，而光催化是指使用光子直接激发无机半导体物质，使其催化所需反应的过程。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功率。光系统I （PSI）是一种生物分子组装，用于将NADP+最终光合还原为NADPH，这是一种为生命系统代谢提供动力的还原性辅助因子。近年来，人们对PSI/生物催化酶融合蛋白的工程设计感兴趣，以利用PSI的光催化能力用于新型还原性生物光催化5。值得注意的是，PSI已经有效地与氢化酶融合，产生高度还原性的蛋白质融合，能够用光子作为电子源催化氢+还原为单质氢6。目的：证明PSI可以作为开发具有生物技术价值的新型光驱动酶的平台。生物工程一系列光系统I/还原酶（PSI/RE）嵌合酶。应用和开发定向进化方法来增强PSI/RE融合的对接、底物特异性、能量学和/或动力学。一系列还原酶将与来自Synechocystis sp. PCC68036的PSI的PsaC亚基融合，产生PSI/RE嵌合体并进行定向进化。感兴趣的还原酶将包括工业主要酶，如细胞色素p450超家族成员1、酶还原酶群7和还原性脱卤酶8，以及碳固定酶Crotonyl-CoA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体外蛋白质工程的最佳途径，以及为定向进化提出目标区域。不同的PsaC/RE融合文库将使用体外和体内诱变技术产生；易出错PCR，位点饱和诱变，以及我们实验室开发的evolvr衍生方法。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。具体来说，筛查方法将包括希夫试剂；底物、产物或辅因子特异性分光光度法13,14；或光活性PSI检测。这项研究将需要对聚囊藻进行基因改造。如果将改造过的蓝藻释放到环境中，这可能构成一个伦理问题；然而，我们实验室的安全和操作程序是符合转基因细菌工作的。

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.