EAGER: Structure-based Engineering of A Diol Dehydratase towards Dehydrating 1,2,4-Butanetriol

对天然酶和生物合成途径的智能表征和了解使酶和全细胞生物催化方法得以发展，用于生产高价值的化学品，如青蒿酸，以及大宗化学品和燃料，如长链醇。然而，在石化工业中合成的许多有价值和有用的化合物并没有可用的生物催化生产方法。因此，迫切需要具有新性质的新型或工程酶来开发这些新的生物催化过程。1，4-丁二醇是石油化学工业中一种高价值的商品化学品，用于生产塑料、聚酯、纤维、药品、化妆品和农药。它的年市场规模为50亿英镑。亚太地区、欧洲和北美是1，4-丁二醇的前三大市场。传统上，1，4-丁二醇的生产完全依赖以石油为基础的化学过程，这些过程具有非常高的能量投入，并且不环保。鉴于对1，4-丁二醇的高需求和对环境的关注，人们非常希望使用可再生资源的替代生产方法，并具有很大的实用价值。酶工程是一种强大的使能技术，已被用于开发许多重要工业应用的新型生物催化剂。它具有高风险的性质，因为很难预测需要多长时间才能找到最佳的突变体。这可能会很快，也可能需要几年时间。然而，可以通过创建和筛选可信的突变体库来管理风险。佐治亚大学的严亚军教授在酶工程方法方面拥有丰富的经验，从而能够生产高价值的商品化学品。他建议进行二醇脱水酶工程的工作，以1，2，4-丁三醇为原料生产高价值的商品化学品1，4-丁二醇。使用二醇脱水酶生产1，4-丁二醇以前从未被提出、研究或开发过。因此，这一热切的提议将具有很强的创新性。在这个急切的提案中，将使用一种合理的酶工程方法来创建一个小型且可信的酶突变体库。二醇脱水酶的结构模型将被用来为文库设计开发假设。在通过对接研究确定催化口袋中最关键的氨基酸残基对酶底物专一性和活性负责后，将在这些位置进行饱和突变以创建突变体库。PI将使用基于厌氧生长的筛选方法来筛选改良突变体的文库。任何对1，2，4-丁三醇表现出新的或改进的活性的突变体都将被测序以确定突变并测定其体外和体内的动力学参数，识别出的有益突变将被纳入结构模型以建立指导催化口袋重建的设计原则。设计原则将考虑三个基本因素：底物对接的催化口袋体积，底物结合的催化口袋中氨基酸残基的电荷和疏水作用，以及底物与电子转移催化位置的相互作用。这些发现将为酶的合理设计提供有价值的信息。更广泛的影响热切提案中的研究工作将是高回报的，因为它将导致开发新的酶突变株，这些酶突变株可用于通过全细胞生物催化从可再生资源中生成1，4-丁二醇，并带来变革性的变化。此外，该研究工作将在分子水平上对二醇脱水酶的催化机理提供新的见解，促进合成有用的酶突变体的更智能合理的重新设计，具有重要的科学意义。拟议的研究将通过为本科生和研究生，特别是来自少数族裔和代表性不足的群体的本科生和研究生创造跨学科的培训和研究机会，进一步加强和多样化佐治亚大学(UGA)生化工程项目的研究和教育。这项拟议的研究还将利用UGA的Young Dawgs计划来教育和培训当地高中的学生，以激发他们对科学和工程的兴趣和热情。

Abstract#1349499Yan, YajunIntellectual Merit Characterization and understanding of natural enzymes and biosynthetic pathways have allowed enzyme and whole-cell biocatalytic approaches to be developed for the production of high-value chemicals such as artemisinic acid, and bulk chemicals and fuels such as longer chain alcohols. However, many valuable and useful compounds that are synthetically derived in the petrochemical industry do not have biocatalytic production approaches available. As a consequence, there is an urgent need for novel or engineered enzymes possessing novel properties to develop these new biocatalytic processes. 1,4-butanediol is a high value commodity chemical in the petrochemical industry for the production of plastics, polyesters, fibers, medicines, cosmetics, and pesticides. It has an annual market of 5 billion pounds. Asian-Pacific, Europe, and North America are the top 3 markets for 1,4-butanediol. Traditionally, 1,4-butanediol production completely relies on petroleum-based chemical processes, which have very high energy input and are not environmentally friendly. Given the high demand for 1,4-butanediol and environmental concerns, alternative production approaches from renewable sources are highly desired and of great practical value. Enzyme engineering is a powerful enabling technology which has been used to develop novel biocatalysts for many important industrial applications. It has the nature of high risk, since it is hard to predict how long it will take to find the optimal mutants. It could be quick or take a few years. However, the risk can be managed by creating and screening creditable mutant pools. Professor Yajun Yan at the University of Georgia has significant experience in approaches to enzyme engineering to enable the production of high value commodity chemicals. He proposes work on the enzyme engineering of a diol dehydratase for the production of the high value commodity chemical 1,4-butanediol from 1,2,4-butanetriol. The use of diol dehydratase for 1,4-butanediol generation has never been proposed, investigated, or exploited before. Therefore, the EAGER proposal will be highly innovative. In this EAGER proposal, a rational enzyme engineering approach to create a small and creditable enzyme mutant library will be used. A structural model of the diol dehydratase enzyme will be used to develop hypotheses for the library design. After identifying the most critical amino acid residues in the catalytic pocket that are responsible for the enzyme substrate specificity and activity by a docking study, saturated mutagenesis at these positions will be performed to create the mutant library. The PI will use the anaerobic growth based screening method to screen the library for improved mutants. Any mutants exhibiting novel or improved activity towards 1,2,4-butanetriol will be sequenced to determine the mutations and assayed to determine their kinetic parameters in vitro and in vivo.The identified beneficial mutations will be incorporated into the structure model to establish a design principle to guide catalytic pocket reconstruction. The design principle will consider three basic factors: catalytic pocket volume for substrate docking, charge and hydropathy of amino acid residues in the catalytic pocket for substrate binding, and interaction between substrates and catalytic sites for electron transfer. The findings will provide valuable information for enzyme rational design in general. Broader Impacts The research work in the EAGER proposal will be high-payoff since it will lead to the development of novel enzyme mutants that can be used for the generation of 1,4-butanediol from renewable sources via whole cell biocatalysis and bring transformative changes. In addition, the research work will provide new insights into the catalytic mechanism of diol dehydratase on a molecular level and promote more intelligent rational redesign of synthetically useful enzyme mutants, which are of great scientific significance. The proposed research will further strengthen and diversify the research and education of the BioChemical Engineering Program at the University of Georgia (UGA) by creating interdisciplinary training and research opportunities for undergraduate and graduate students, especially those from minorities and underrepresented groups. The proposed research will also use the Young Dawgs Program at UGA to educate and train the students from local high schools to inspire their interests and enthusiasm in science and engineering.