Research

Research in the Enzyme Engineering group focuses on three main areas related to enzymes:

1) Engineering of Enzymes for Efficient Production of Value-added Chemicals from Sustainable Resources:

Fatty Acid Hydratases for Production of Hydroxy Fatty Acids: Oxo-functionalization of hydrocarbons is a key reaction in production of precursors for manufacturing of plastics and various other materials. Lipids are sustainable feedstock for industry, but often require oxo-functionalization on their hydrocarbon backbones. Fatty acid hydratases catalyze addition of water to double bonds in long-chain unsaturated fatty acids, forming hydroxy fatty acids. In addition to being starting materials for chemical industry, hydroxyl fatty acids have beneficial health properties. In a Novo Nordisk Foundation funded project led by Prof. Zheng Guo, we are currently working on engineering of hydratases for a broader substrate promiscuity and activity, in collaboration with researchers from DTU, Denmark and from Kyoto University, Japan. Through rational design and semi-rational engineering approaches, we aim to generate hydratase variants that can hydrate uncommon double bond positions as opposed to their native counterparts. This way, we want to generate authentic molecules with various beneficial properties and want to show, through pilot scale production, the feasibility of microalgae as a convenient feedstock for industrial production of hydroxyl fatty acids.

Fatty Acid Hydratases for Production of Hydroxy Fatty Acids. Figure: Bekir Eser, AU.

Fatty Acid Decarboxylases for Production of Drop-in Biofuels and Platform Chemicals: Sustainable and green production of biofuels from renewable sources has attracted great attention due to the limited availability and adverse environmental impact of petroleum. Through engineering and optimization of the natural reactions of recently discovered fatty acid decarboxylases, we aim to provide efficient and green routes to drop-in biofuels (biofuels similar to fossil fuel in chemical makeup) and platform chemicals. Furthermore, we plan to reprogramme the same enzymes in such a way that they catalyze useful synthetic radical based reactions other than their native reactions. This will lead to a greater repertoire of valuable chemicals produced by these enzymes and will open up a new horizon in non-natural chemistry catalyzed by enzymes in general. Finally, we want to demonstrate feasibility of our enzymatic synthesis strategies through pilot-scale production using sustainable fatty acid feedstock.

Fatty Acid Decarboxylases for Production of Drop-in Biofuels and Platform Chemicals. Figure Bekir Eser, AU.

Polymethoxyflavone Demethylases – Enzymes with High Potential for Synthesis of Valuable Natural Product Derivatives: Polymethoxyflavones (PMFs) are an important class of flavonoids with various biological activities such as anti-cancer, anti-inflammation and anti-allergic. Human gut microbiota further converts PMFs to various demethylated intermediates that can have unique biological activities. We want to explore potential of the cobalt-dependent enzyme that carry out O-demethylation reaction, through mechanistic investigations and enzyme engineering. For this project, we collaborate with researchers from Chung-Ang University in South Korea.

Demethylation of Polyymethoxyflavones by gut bacteria. Adapted from Kim, Mihyang, Nayoung Kim, and Jaehong Han. Journal of agricultural and food chemistry 61.51 (2014): 12377-12383. — Demethylation of Polymethoxyflavones by gut bacteria. Adapted from Kim, Mihyang, Nayoung Kim, and Jaehong Han. Journal of agricultural and food chemistry 61.51 (2014): 12377-12383.

2) Discovery and characterization of new enzymes and enzymatic pathways:

With the advances in biological sciences over the last couple of decades, it is now possible to access total genome and proteome information of various organisms. This enables researchers to perform bioinformatics search to find known metabolic pathways in a new organism or to find a new pathway that has been unknown in any organism before. There are increasing numbers of protein encoding genes that has been identified and sequenced, but the functions of the translated proteins from many of these genes are either unknown, or poorly predicted, or even sometimes falsely annotated. Using bioinformatics approaches coupled with experimental enzymology studies, many of these genes can be identified with new functions or with functions only known in certain organisms. Exploration of their reactions will lead us to discover potential catalytic candidates for industrial and/or medical applications.

3) Mechanistic Enzymology:

A clear understanding of the chemical and kinetic mechanisms of enzymatic transformations is important for engineering and establishment of production strategies with enzymes. In order to engineer enzymes by rational design and to find the optimum conditions for efficient production, characteristics of the enzyme such as kinetic behavior, inhibition patterns, substrate scope, cofactor requirement, rate-limiting step and optimum pH/temperature should be investigated in detail. We use methods including steady-state and transient-state enzyme kinetics, various types of spectroscopy, isotope-labeling experiments and site-directed mutagenesis to reveal the mechanism of the enzymes that we aim to engineer. The results from such experiments present us important guidelines for rational protein design as well as for fully exploring natural and non-natural capabilities of the enzymes. Moreover, such mechanistic information also contributes to current scientific knowledge on the working principles of enzymes.

Mechanistic Enzymology. Figure: Bekir Eser, AU.

RESEARCH TOOLS

In our research, we use various experimental approaches including common molecular biology techniques (gene cloning, PCR, plasmid purification, site-directed mutagenesis etc.), protein expression and purification, general protein characterization techniques, rational and random protein engineering methods, enzyme kinetics, enzyme activity analysis and assay design, product analysis and identification by chromatographic and spectroscopic methods (GC-FID, HPLC, GC-MS, NMR). Our methodology covers a broad range from molecular biology and bioinformatics to chemistry and biophysics.