Engineering Photosystem I for Light-Driven Biocatalysis

还原催化是许多工业和制药过程中的中心元素，包括生物活性化合物的功能化1和烯烃的还原2，但在标准条件下，化学催化通常是不稳定的或功能惰性的。这导致了巨大的能量和资源消耗，以保持正确的温度和压力环境，使这些反应取得成果。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2和光催化3领域。生物催化是指使用新型酶来催化反应，而光催化是一种利用光子直接激发无机半导体物种，使其能够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获得更大的还原能力。光系统I(PSI)是一种生物分子组件，负责将NADP+最终光合作用还原为NADPH，NADPH是生命系统中为新陈代谢提供动力的还原辅因子。近年来，人们对设计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生物光催化产生了兴趣。值得注意的是，PSI已经与氢酶有效地融合在一起，产生了高度还原的蛋白质融合，能够利用光子作为电子源催化H+还原为元素氢6。目的证明PSI可以作为一个平台来开发具有生物技术意义的新型光驱动酶。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，以增强PSI/RE融合的对接、底物专一性、能量学和/或动力学。研究一系列还原酶将与聚球藻PSI的PSAC亚单位融合。PCC68036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食，如细胞色素P450超家族成员1、烯还原酶组7和还原脱卤酶8，以及碳固定酶巴豆酰-辅酶A羧基酶/还原酶(CCR)9和一系列工业感兴趣的醛/醇脱氢酶10。在电子计算机中，融合前和融合后的酶结构的预测将被用于提供体外蛋白质工程的最佳途径，以及为定向进化提供目标区域的建议。将使用体外和体内突变技术产生不同的PSAC/RE融合文库；容易出错的PCR、位置饱和突变和我们实验室开发的EvolvR衍生方法11。为此，将利用吸收、荧光探针和比色法，开发用于筛选和选择光驱动嵌合体功能的酶特异性方法。具体来说，筛选方法将包括席夫试剂12；底物、产物或辅因子特定的分光光度计13、14；或光活性PSI检测15。伦理考量这项研究将有必要对聚球藻进行基因改造。如果转基因蓝藻被释放到环境中，这可能会构成一个伦理问题；然而，我们实验室的安全和操作程序符合使用转基因细菌的要求。

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.