Abstract: Over 15,000 NAD(P)H-dependent enzymes discovered in Nature represents a rich yet largely untapped resource for catalyst development, with many of these enzymes outperforming any man-made catalysts. To meet the catalytic needs in efficiently converting renewable resources into fuels and chemicals, we established a range of platform technologies to configure these enzymes: First, we developed multiple ultrahigh-throughput (~109 candidates per round of selection), universal, in vivo selection platforms which use cell growth as an easy readout of NAD(P)H-dependent enzyme's activity. Using these selection platforms, we have achieved, through just a single round of selection, remodeling of 4-hydroxybenzoate hydroxylase (PobA) to accept a nonnative substrate for natural product biosynthesis, improving the coupling efficiency of cytochrome p450 BM3 for environmental pollutant degradation, and switching the cofactor specificity and enhancing the thermal stability of an industrially important enzyme cyclohexanone monooxygenase (CHMO). Second, we developed a non-canonical redox cofactor system based on nicotinamide mononucleotide (NMN+), which is a much cheaper alternative to NAD(P) in vitro and operates in an orthogonal fashion to NAD(P) in vivo. We demonstrate that this system can be used to support diverse redox chemistries with high robustness, to specifically deliver reducing power in both whole-cell and cell-free biotransformation, and to shift the redox reaction equilibrium on demand. We developed two growth-based selection platforms to evolve enzymes that can oxidize or reduce NMN+, which allowed deep searching of the protein sequence space that give rise to the general design principles. These technologies will not only enable facile control of redox reactions in Nature, but also pave the way for readily expanding what Nature can do to manufacture green chemicals for human society.
Bio: Dr. Han Li is an Associate Professor and Vice Chair of Chemical and Biomolecular Engineering at University of California, Irvine. She obtained her B.S in Biological Sciences from Tsinghua University, China in 2008, and her Ph.D. with Dr. James Liao at University of California, Los Angeles in 2013, and conducted postdoctoral study with Dr. Peter Schultz at The Scripps Research Institutes, San Diego in 2013-2015. Her group pioneers artificial cofactor technology and redox balance-based enzyme evolution. She has been recognized by NSF CAREER Award, NIH Director's New Innovator Award, Sloan Research Fellowship, and Finalist of Blavatnik national awards for young scientists.
