Molecular mechanisms underlying evolution of new functionWhat is the molecular mechanisms for evolution of new functions? In order to gain detailed insights into enzyme evolution, we employ intervdeceplinary approaches, experimental (or laboratory) evolution, bioinformatics (ancestral reconstruction), high-throughput mutational analysis and diverse biochemical and biophysical characterizations. Experimental evolution allows us to evolve enzymes towards new activities. All parameters in experimental evolution, e.g., mutation rate, mutation type, selection pressure and the environment, are tunable, thus we can controllably test diverse aspects of molecular evolution. The evolutionary intermediates that are obtained represent a molecular “fossil record”, and enable us to explore the step-by-step evolution of molecular function. We characterize the “molecular fossils” that are generated by laboratory evolution to reveal the molecular mechanisms of the functional transition. Using biochemical, structural, genetic and computational techniques, we study how structural change is associated with functional transition – we are particularly interested in understanding how distant mutations can remotely affect the active site architecture (e.g., protein dynamics) and affect enzyme activity. We have explored diverse systems for understanding the mechanisms underlying an evolutionary transition between distinct enzymatic activities, e.g., phosphotriestrase vs arylesterase, lactonase vs phopsphotriesterase, beta-lactamase vs phosphonate monoester hydrolase. |
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Epistasis and evolutionary constraints“Epistasis”, i.e., interactions between mutations, is a fundamental phenomenon that constrains, limits and stalls evolutionary processes. Understanding epistasis is essential for the effective design, engineering and evolution of new enzymes and proteins in the laboratory. How prevalent is epistasis in protein evolution? Should be care about epistasis? How does epistasis prevent a evolutionary trajectory from reaching a high fitness peak? Epistasis is also deeply related to many long-standing evolutionary questions, e.g., “Is evolution reversible?” and “Is evolution repeatable?”, “how evolution is contingent on evolutionary history?”. We use diverse approaches, experimental evolution, high-throughput mutational characterizations and statistical approaches to quantify epistasis. Furthermore, we characterize evolutionary trajectories in detail using various techniques to study the molecular basis underlying these constraints. |
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Evolution of antibiotic resistanceThe emergence and dissemination of antibiotic resistance is one of the major threats to human health. While our combat against antibiotic resistance has only occurred in the last century, most antibiotic resistance genes are ancient, and originated more than billion years ago. Thus, there are numerous “antibiotic resistome” exist among environmental microbes, and they are continuously evolving to adapt our new generations of antibiotics. An efficient system to predict and understand the evolution of antibiotic resistance would provide us with an advantage in the battle against resistant pathogens. To this end, we employ experimental evolution and high-throughput mutational analysis (called deep mutational scanning, or DMS)to examine the evolutionary potential (evolvability) and molecular basis of adaptation in antibiotic resistance genes. β-lactamases are one of the major bacterial weapons against β-lactams, i.e., these are the enzymes that result in bacterial resistance to common antibiotics such as ampicillin. We also perform comprehensive large scale bioinformatics to unveil unexplored antibiotic resistome in environmental microbes. We aim at combining those massive bioinformatics and experimental data to develop predictive models, which can predict evolutionary trends of antibiotic resistance in the future. |
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Comprehensive bioinformatics and enzyme characterizationsAn enzyme superfamily is comprised of enzymes that share a common fold and catalytic machinery but can catalyze distinct chemical reactions. This exemplifies the power of molecular evolution, as members of a superfamily share structural and mechanistic features, and it has been postulated that they divergently evolved from a common ancestral enzyme. How do enzymes evolve such functional diversity? In order to obtain a global view of evolution within enzyme superfamilies, we perform a large-scale, comprehensive bioinformatics (e.g., Sequence Similarity Networks (SSNs)), and phylogenetic analysis. We also perform a large-scale experimental analysis of enzymes across the superfamily. In particular, we are interested in revealing the functional connectivity via enzyme promiscuity between the distinct functions of a superfamily. We aim to unveil the sequence-structure-function relationships, and understand molecular blueprints for enzyme functions. |
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Evolution of metabolic pathways and microbial communityHow do metabolic pathways with multiple enzymes evolve? Do multiple enzymes have to emerge simultaneously within a single organism? Or, is it possible that multiple organisms with unique functional ability, e.g., microbial community, can provide a foundation of multi-enzyme metabolic pathways? We take a synthetic biology approach to study a xenobiotic degradation pathway to degrade organophosphate pesticides that emerged in bacteria in the last century. In particular, we reconstruct the metabolic pathway based on microbial community and test the evolutionary dynamics of the new metabolic pathways. |
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Single molecule kinetics for enzyme promiscuity and evolutionEnzymes are generally thought to form a unique 3D structure for their catalysis. However, in the atomic level, they exhibit distinct multiple conformational substates, which underlie the existence of functional substates within a population. So true understanding for enzyme behaviour requires studies in the single molecule level. We are excited to adopt “single molecule enzyme kinetics” in our lab. Using a micro-chamber system, now we can observe behaviours of >10,000 single enzyme molecule under the microscope. This allow us to observe the distribution of functional substates within a single enzyme population. We will use this cutting edge-technology to unveil the molecular mechanisms for enzyme promiscuity, multifunctionality and evolution. |
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Enzyme discovery and engineeringWe apply bioinformatics and experimental approaches towards the discovery and engineering of new enzymes. We are currently engineering enzymes for antibacterial and anti-biofilm reagents, e.g., enzymes that degrade bacterial cell-to-cell signalling molecules (Quorum sensing molecules) and disrupt the formation of biofilms. |
