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.
