Huimin Zhao

Research

Protein Engineering and Metabolic Engineering

Research in the Zhao group is focused on protein engineering and metabolic engineering. The overall research theme is to use directed evolution in combination with rational design to create proteins, receptors, biosynthetic pathways, and whole cells with improved or novel functions, followed by detailed biochemical and biophysical characterizations. There are dozens of ongoing projects in the lab, which can be generally grouped into four different areas as described below. It should be noted that although the following projects are all application-driven, we are also very interested in addressing fundamental questions related to protein structure-function relationship, enzyme catalysis, molecular recognition, gene regulation, and immunology.

Industrial Biotechnology and Bioenergy

We successfully used directed evolution and rational design approaches to develop a novel phosphite dehydrogenase based NAD(P)H regeneration system by (1) improving the enzyme activity toward NADP by 1000-fold, (2) increasing the overall activity by 6-fold, and (3) increasing the thermostability by more than 22,000-fold. The resulting technology was patented and licensed to BASF and Biocatalytics. Based on this new cofactor regeneration method, we are developing new in vitro and in vivo methods for cost-effective production of xylitol, one of the twelve platform chemicals that the Department of Energy has identified for the emerging biorefinery industry. In addition, we are interested in converting renewable feedstock such as corn stovers into biofuels including ethanol, butanol, and other long-chain alcohols. Using a combination of protein engineering, metabolic engineering, bioinformatics, and functional genomics, we attempt to address key roadblocks in fermentative production of chemicals and biofuels from renewable feedstock, including limited range and poor efficiency of sugar utilization, relatively low product yield, productivity and titer, and low tolerance level to products and/or substrates.

Drug Discovery and Development

We are using protein and metabolic engineering tools to overproduce phloroglucinol, an important pharmaceutical intermediate in E. coli. Several biosynthetic pathways and enzymes such as type I polyketide synthases and type III polyketide synthases have been characterized and engineered. In addition, novel enzymes involved in arylamine oxygenation have been discovered and characterized using various biochemical and biophysical methods such as HPLC, EPR, LC-MS, and protein crystallography. In addition, in collaboration with the Metcalf, van der Donk, Kelleher, and Satish groups at University of Illinois, we are characterizing and engineering a new class of natural products, so-called phosphonic acids, many of which show antimicrobial, anticancer, and fungal and herbicidal activities. We recently cloned the gene cluster involved in the synthesis of fosfomycin, an FDA-approved antibiotic, and are now attempting to overproduce it in both E. coli and yeast. We are also interested in another phosphonic acid compound, FR-900098, an anti-malarial agent, among many others. In addition to overproducing these antibiotics, we are also using combinatorial biosynthesis and directed evolution approaches to generate novel derivatives of phosphonic acids with the hope of discovering new antibiotics with improved biological function.

Synthetic Biology and Gene Therapy

We are using directed evolution and rational design approaches to create a variety of transcription factor based gene switches and gene circuits to precisely control the gene expression levels in mammalian cells and animals. Such gene switches and circuits are invaluable tools for gene therapy, tissue engineering, functional genomics, therapeutic protein production, and stem cell engineering. In collaboration with world-leading scientists including Drs. John Katzenellenbogen, William Helfrich, Pierre Chambon, and Daniel Metzger, we are evaluating these tools in both mammalian cells and transgenic animals and applying them to address many different biomedical challenges. In addition, we are creating homing endonuclease based gene scissors that can introduce specific double strand breaks in a target gene. Such tools represent a new therapeutic regime for gene therapy. Two genetic diseases of particular interest are cystic fibrosis and sickle cell anemia.

MHC Engineering and Immunotherapy

We are applying protein engineering and yeast cell surface display to create yeast based artificial antigen presenting cells for analysis and engineering of human class II major histocompatibility complex (MHC) molecules for diagnostic and therapeutic applications. Based on this new system, we recently developed a new method for identifying CD4+ T-cell epitopes, which is a critical but often difficult limiting step for pathogenesis studies and a wide range of immunological and clinical applications. We are now further optimizing this system and extending it to other biomedical applications such as vaccine development, tumor antigen identification, T-cell staining, and treatment of autoimmune diseases.

Publications

T. Johannes, R. Woodyer, and H. Zhao. "Efficient Regeneration of NADPH using an Engineered Phosphite Dehydrogenase," Biotechnology and Bioengineering, 96, 18-26 (2007).

H. Zhao and W. Zha, "In vitro 'Sexual' Evolution through the PCR-based Staggered Extension Process (StEP)," Nature Protocols, 1, 1865-1871 (2006).

R. Woodyer, Z. Shao, P. M. Thomas, N. L. Kelleher, J. A. V. Blodgett, W. M. Metcalf, W. A. van der Donk, and H. Zhao, "Heterologous Production of Fosfomycin and Identification of the Minimal Fosfomycin Biosynthetic Cluster," Chemistry & Biology, 13, 1171-1182 (2006).

W. Zha, S. Rubin-Pitel, and H. Zhao, "Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase from Pseudomonas fluorescens," J.Bio. Chem., 281, 32036-32047 (2006).

M. Simurdiak, J. Lee, and H. Zhao, "A New Class of Arylamine Oxidases: Evidence that p-Aminobenzoate N-oxidase is a Diiron Enzyme and Further Mechanistic Studies," ChemBioChem, 7, 1169-1172 (2006).

T. Johannes and H. Zhao, "Directed Evolution of Enzymes and Biosynthetic Pathways," Current Opinion in Microbiology, 9, 261-267 (2006).

J. Lee and H. Zhao, "Mechanistic Studies on the Conversion of Arylamines into Arylnitro Compounds by Arylaminopyrrolnitrin Oxygenase: Identification of Intermediates and Kinetic Studies," Angew. Chem. Int. Ed., 45, 622-625 (2006).

Z. Chen and H. Zhao, "A Highly Sensitive Selection Method for Directed Evolution of Homing Endonucleases," Nucleic Acids Research, 33, e154 (2005).

K. Chockalingam, Z. Chen, J.A. Katzenellenbogen, and H. Zhao, "Directed Evolution of Specific Receptor-Ligand Pairs for Use in the Creation of Gene Switches," Proc. Nat. Acad. USA, 102, 5691-5696 (2005).

Z. Chen and H. Zhao, "Rapid Creation of a Novel Protein Function by in vitro Co-evolution," J. Mol. Bio., 348, 1273-1282 (2005).

Awards

• Helen Corley Petit Scholar, 2006
• DuPont Young Investigator Award, 2005
• Xerox Award for Faculty Research, UIUC College of Engineering, 2005
• Beckman Fellow, UIUC Center for Advanced Study, 2005-2006
• Excellence in Teaching Award, UIUC School of Chemical Sciences, 2004
• NSF CAREER Award, 2004
• Collins Scholar, UIUC Academy for Excellence in Engineering Education, 2001
• Special Recognition Award, Dow Chemical Company, 1999, 2000

Highlights