Spore-Based Designer Enzyme Cascade Biocatalysts

摘要：通过化学转化创造的增值产品，如精细化学品和药品，极大地提高了我们的生活质量。这些化学转化过程中有许多都使用催化剂。这些催化剂可以是无机的，也可以是生物的。酶催化剂具有高产率、高选择性、高产品纯度、常温常压、pH适中等优点，将被广泛应用于这些化学转化过程。然而，许多生物催化反应涉及昂贵的辅酶或辅因子，它们的回收对于这些过程的成本效益至关重要。事实证明，这是一个困难或昂贵的过程步骤，从而限制了获得使用酶催化剂的优势的能力。加州大学河滨分校的首席研究员辛戈和阿肖克·马尔昌达尼着眼于自然界中的细胞反应，以开发一种绕过这个问题的方法。受自然界中为避开不利的热力学和动力学而出现的多酶级联中的底物沟道现象的启发，PI将探索在孢子表面开发模块化设计的生物催化平台，其中酶级联通过可调的化学计量比在空间上组织，以实现高效的辅因子再生。该酶体系易于生产和重复使用，稳定性高。该系统的模块化性质将允许轻松插入所需酶的基因并控制表面的化学计量比。这一合作研究项目具有重要意义，因为它将导致为设计师生物催化剂开发一种新的强大的模块化平台，以满足化学品和药品制造的需求。可以很容易地预见到许多应用。改进的催化剂和工艺将提高美国的技术竞争力。总而言之，这项研究的好处将支持许多精细化学品和药品的高效、经济和绿色工程生产。此外，私营部门还计划开展活动，通过增加妇女和代表性不足的少数群体的参与，培养一支具有全球竞争力和多样化的STEM劳动力队伍。加州大学河滨分校是加州大学所有校园中拉美裔学生人数最多的少数族裔服务机构。研究人员计划聘请少数族裔研究生和本科生作为这个项目的研究助理。研究人员还计划开设新的课程，并与当地一所中学合作，建立一个名为生物催化清洁燃料的互动科学项目。大多数参与特殊化学合成的氧化还原酶利用昂贵的吡啶核苷酸作为催化的辅助因子。这些酶催化工艺在辅因子回收方面存在缺陷，限制了总周转率和生产率。本研究的目标是开发一种模块化设计型生物催化剂平台，用于高效的辅因子再生。受自然界中观察到的底物通道现象和其他工程多酶级联和微型纤维素体研究的启发，支架-粘附素-Dockerin系统将用于构建空间有序的多酶复合体，旨在高效地再生辅因子。这种酶复合体将由位于附近的所需化学计量比的产生和再生脱氢酶组成，以允许氧化辅因子从产生的脱氢酶向再生脱氢酶输送，反之亦然，从而解决再生问题。由于其对极端温度、pH、溶剂、湿度和辐射的强大抵抗力，细菌孢子将作为酶级联的表面展示。对典型的酮还原为醇的合成反应进行了各种对照和参考实验，并展示了辅因子在水和非水介质中的酶偶联再生。PIS计划进行广泛的表征和催化性能评估。这些信息将被公布，并可供其他生物催化应用的研究人员使用。

Abstract: The creation of value-added products such as fine chemicals and pharmaceuticals by chemical transformations has resulted in significant improvements in the quality of life we have been enjoying. Many of these chemical transformation processes use catalysts. These catalysts may be inorganic or biological in nature. Enzyme catalysts would be widely utilized to perform these chemical transformation processes, as they frequently offer advantages of high yield, high selectivity, high product purity, along with operation at ambient temperature and pressure in aqueous environment at moderate pH. However, many biocatalytic reactions involve expensive co-enzymes or co-factors and their recycling is essential for the processes to be cost-effective. This turns out to be a difficult or expensive process step, thereby limiting the ability to gain the advantages of using enzyme catalysts. Principal investigators Xin Ge and Ashok Mulchandani from the University of California Riverside looked to cellular reactions in nature to develop an approach to circumvent this issue. Inspired by the substrate channeling phenomena seen in multi-enzyme cascades in nature for circumventing unfavorable thermodynamics and kinetics, the PIs will explore the development of a modular designer biocatalyst platform on the surface of spores, where enzyme cascade is spatially organized with tunable stoichiometry to achieve highly efficient cofactor regeneration. The enzyme system is easy to produce and reuse, and has high stability. The modular nature of the system will allow easy insertion of the genes of the desired enzymes and control of the stoichiometric ratios on the surface.This collaborative research project is significant as it will lead to development of a novel robust modular platform for designer biocatalysts to address the needs of chemicals and pharmaceuticals manufacturing. A number of applications are readily envisioned. The improved catalysts and processes will increase US technological competitiveness. Collectively, the benefits from this research will support efficient, economical and green engineering production of many fine chemicals and pharmaceuticals. In addition, the PIs plan activities which will develop a globally competitive and divergent STEM workforce through the increased participation of women and underrepresented minorities. UC Riverside is the minority serving institution with the largest Hispanic student population among all UC campuses. The investigators plan to hire minority graduate and undergraduate students as research assistants for this project. The investigators also plan new curriculum efforts and are collaborating with a local middle school to establish an interactive science program titled Bio- catalysis for clean fuels. Most oxidoreductase enzymes involved in specialty chemical synthesis utilize expensive pyridine nucleotides as cofactors for catalysis. These enzyme catalytic processes have shortcomings in terms of cofactor recycling that limit total turnover number and productivity yields. The goal of the proposed research is to develop a modular designer biocatalyst platform for highly efficient cofactor regeneration. Inspired by the substrate channeling phenomena observed in nature and other studies of engineered multienzyme cascades and mini-cellulosomes, the scaffoldin- cohesin - dockerin system will be used to build a spatially organized multienzyme complex designed for highly efficient cofactor regeneration. This enzyme complex will consist of proximally located producing and regenerating dehydrogenases in desired stoichiometry on a selected surface to allow channeling of oxidized cofactor from the producing dehydrogenase to the regenerating dehydrogenases and vice versa, solving the regeneration problems. Because of their formidable resistance to extremes of temperatures, pH, solvents, humidity and radiations, bacterial spores will serve as the surface display for the enzyme cascade. Various control and reference experiments will be carried out for the typical synthesis reaction of ketone reduction to alcohol, and the enzyme coupled regeneration of the cofactor will be demonstrated in both aqueous and nonaqueous media. Extensive characterization and catalytic performance assessments are planned by the PIs. This information will be published and available for investigators of other biocatalytic applications.