The complex interactions between humans and the rest of the biosphere have created some of our most challenging global problems in human history such as energy sustainability, severe pollutions, and emergence or re-emergence of old and new epidemics and diseases. Research in our laboratory is focused on the development of next generation synthetic biology tools in addressing these key global problems. Proteins are the most versatile among the various biological building blocks. However, the strength of proteins - their versatility and specific interactions - also complicates and hinders their systematic design and engineering. Our lab has been interested in exploiting the modular nature of protein domains to design synthetic complexes that can perform new biological functions across different length scales. By adding logical and stimuli responsive components into the design, smart protein complexes can be created to sense and adapt to the constantly changing cellular environments. We are currently working on several projects in connecting exchangeable protein domains into functional devices for synthetic biology applications in biocatalysis, biosensing, and therapeutics.


Current Research

I supervise a group composed of 2 postdoctoral researchers and 12 Ph.D. students. A brief description of all the current research activities is summarized below.


1. Synthetic extracellular sensing circuit by intein-mediated reconstitution of yeast mating factor (NSF)

Increasing demands for alternative fuels such as bioethanol has driven the development of more efficient processes. In particular, the use microbial consortia is a promising approach to dramatically reduce costs and to improve fuel production directly from renewable biomass. However, regulation and coordination of different populations within the consortia remain challenging since currently available intracellular sensing circuits are incapable of responding to the two key substrates, cellulose and hemicellulose, that are presented exclusively in the extracellular medium.  To meet these challenges, we present a new synthetic extracellular sensing circuit to control yeast behaviors by reconstitution of extracellular protein sensors. These synthetic fusion proteins are designed to trigger split intein-mediated reconstitution of functional α-factor peptide analogues in response to nutrient conditions. The reconstituted α-factors bind onto Ste2p, a native yeast G protein-coupled receptor, and activate the yeast mating response and transcriptional induction. By integrating this native mating signaling pathway with heterologous gene expression cassettes, we can control timing and level of induction and direct phenotypic behaviors by applying our split intein fusion proteins in yeast cultures. This synthetic control strategy will enable regulation of yeast functions in a wide range of applications, including consolidated bioprocessing.



2. DNA guided assembly of enzyme cascades for biocatalytic fuel cell applications (NSF)

Enzymatic fuel cells have received considerable attention because of their potential for direct conversion of abundant raw materials to electricity. The use of multi-enzyme cascades is particularly attractive as they offer the possibility of achieving a higher current density by the sequential oxidization of fuels. Efficient substrate and electron channeling are two of the most important bottlenecks in improving the power output of multi-enzyme fuel cells. The overall goal of this work is to investigate the use of a genetically controlled, DNA-based modular approach for the self-assembly of a multi-enzyme cascade for enhanced substrate and electron channeling. Because of the modular nature of the design, we believe this framework will have a huge impact on the assembly of many multi-enzyme cascades useful for biocatalysis and fuel cell applications.


Zinc-finger proteins (ZFPs) are DNA binding proteins that are composed of three subunits with each recognizing a specific three base-pair (bp) sequence. By taking advantage of the modularity of the individual zinc finger protein, multiple zinc finger motifs with nanomolar affinity have been designed to sketch the target sequences from 9 to 45 bp while retaining the exquisite specificity. In a paper published in Chemical Communication, artificial cellulosome structures were created on DNA scaffolds based on zinc finger protein (ZFP)-guided assembly. A synthetic DNA scaffold was used for the site-specific organization of two ZFP-appended proteins (an endoglucanase CelA and a cellulose-binding module CBM from Clostridium thermocellum) into a bifunctional cellulosome structure for enhanced cellulose hydrolysis. These resulting two-component cellulosome structures exhibited enhancement in cellulose hydrolysis compared to the non-complexed mixture depending on the number of CBMs and cellulases assembled. The modular nature of the design allows easy alteration of the number, spacing, and ordering of enzymes assembled, leading to the virtually unlimited combination of artificial cellulosome structures optimized for a given target cellulosic substrate.


3. Engineering Bacterial Outer Membrane Vesicles for New Biotechnology Applications (NSF)

Outer membrane vesicles (OMVs) are protein, lipid, and polysaccharide-based vesicles derived from the outer membrane and periplasmic space of Gram-negative bacteria. It is now firmly established that E. coli can be engineered to incorporate heterologously expressed proteins or plasmid DNA into different compartments of OMVs. Although engineered OMVs with either an antigenic protein or peptide expressed on the surface or in the lumen have been widely used as a delivery vehicle for protein subunit vaccines, very little has been done to fully realize the relatively untapped potential of OMVs by expanding the range and complexity of biomolecular structures that can be functionally produced in their lumens, in their membranes, and on their surfaces. My group are developing tools necessary to assemble synthetic OMVs for cell-specific targeting, gene/protein delivery, vaccine development, cascading enzyme reactions, biosensing and bioremediation.


My group has successfully demonstrated a facile method to create nanoscale enzyme cascades using synthetic bacterial outer membrane vesicles (OMVs) displaying a trivalent protein scaffold. The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes. Our synthetic biology approach excludes the use of any chemical conjugation and can provide a simple platform for generating nanobiocatalysts for highly efficient multi-enzyme reactions. The flexibility in designing scaffolds using a wide range of specific binding domains will permit the virtually unlimited number of functional proteins to be displayed on the OMVs. 


4 Synthetic Methylotrophy to Liquid Fuel (ARPA-E)

We are in the process of engineering Methanosarcina barkeri Fusaro, a methanogenic archaeon with a well-developed genetic toolbox, to enable highly efficient anaerobic methane oxidation and the subsequent conversion to methanol with high efficiency, yield and rate. First, methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis will be used to carryout efficient anaerobic methane oxidation for the activation of methane into CH3-S-CoM. These MCR-expressing strains will be further engineered to grow on methane by regeneration of CoM-S-S-CoB. This will be achieved either by addition of exogenous electron acceptors or by fumarate reductase coupling. The initial step of methane activation will be optimized by creating improved MCR variants by both rational design and directed evolution. Next, we will optimize the conversion of CH3-S-CoM to methanol by characterizing and engineering the key enzyme (MTaABC Methanol:coenzyme M methyltransferase) in the pathway. Gene expression for these pathways will optimized for fast growth on methane and effective methanol production. A series of miniature, high-pressure bioreactors will be constructed to test the engineered strains.


Methanol produced from the anaerobic methane activation will be further utilized by the engineered E. coli strains for butanol production. The approach is to simultaneously use methanol (MeOH) and CO2 to produce n-butanol from an engineered E. coli strain. My group will focus on designing protein scaffolds to improve the efficiency of the pathway and increase the carbon flux by overcoming the thermodynamics barriers of these reactions.



5. Industrial Implementation of Smart Biopolymers for Purification of Biological Products (NSF)

This research will result in a flexible platform for large-scale purification of biological products. The first component of the proposed project will be a new approach for affinity precipitation of mAbs and Fc fusion proteins under milder operating conditions using Z-ELP-E2 nanocages. The second component of the project will develop novel peptide affinity ligands for two classes of non-mAb proteins provided by our industrial collaborator BMS. The affinity peptides will then be employed in affinity precipitation formats to selectively capture the products from challenging feed stocks such as refolded protein pools and post pegylation reaction mixtures. This work brings together Co-PIs with diverse expertise in protein engineering, peptide affinity design, affinity precipitation and downstream bioprocessing to develop entirely new classes of affinity precipitation reagents (Z-ELP-E2 nanocages, ELP-affinity peptides, and polyvalent affinity capture E2-(xAPy-ELP)) which will result in a new integrated platform that will greatly simplify the recovery and purification of biological products.




6. Synthetic multilayer targeting DNA devices for detection of specific cancer indicators and programmed assembly of split yCD for prodrug activation (NSF)

One of the most pressing needs in cancer treatment is to distinguish and treat cancer vs healthy cells. Active targeting of surface markers alone is inadequate and must be merged with additional layers of intracellular signals to provide a higher level of specificity. Advances in synthetic biology have enabled the engineering of new cellular sensors, actuators, and amplifiers toward the creation of clinically relevant “smart drugs” that sense the disease state in a complex cellular environment and actuate an appropriate, localized therapeutic response for treatment. Prior efforts have primarily been focused on implementing therapeutic gene circuits for targeted cancer therapies. However, practical utility of these synthetic devices is limited as multiple genes must be delivered and expressed within the target cell to produce needed detection components. Less complex DNA-based logic devices can potentially bypass this stringent delivery hurdle but the few successful examples reported so far can only be executed in vitro due to the need for a restriction enzyme as part of the activation circuit. Dynamic DNA-gated lock and key devices based on toehold-mediated strand displacement is a new powerful method to create intracellular logic circuits that can be triggered by cancer-specific biomarkers. By combining these dynamic DNA devices with active extracellular targeting and exogenously applied, inactive prodrugs to trigger cell death, a higher degree of specificity for cancer cells can be achieved by creating these multi-layer targeting, sensing, and responsive DNA-based devices. In this proposal, we seek to develop a new generation of synthetic DNA devices based on toehold-mediated strand displacement that can be used for programmable intracellular reconstitution of the yeast cytosine deaminase (yCD), a split suicide enzyme capable of activating the prodrug deaminate 5-fluorocytosine (5-FC) into the toxic product 5-fluorouracil (5-FU). Although the simplicity of the design allows highly adaptable, multi-input capabilities suitable for a range of cancer targets, the feasibility of the approach will be illustrated with a two-input device using two microRNAs (miRNAs) targets, miR-720 and miR-1380, associated with highly invasive inflammatory breast cancer (IBC) cells, as inputs.



7. Design of Multi-Functional SplitCore HBV Capsids for Precisely Controlled Multi-siRNA Delivery in Cancer Therapeutics (NSF)

Virus-like particles are perfectly monodisperse self-assemblies of protein-only subunits that are ideal for therapeutic delivery applications because both capsid surfaces and interiors can be stoichiometrically decorated with moieties for cell targeting and siRNA capture/release. We will exploit the newly discovered SplitCore strategy for the formation of HBV capsids using fragment complementation of the separately expressed N- and C-cores to provide a streamlined approach to attach four unique decorations (3 exterior, 1 interior) to each HBV monomer into a highly modular nanoplatform suitable for customizable delivery and siRNA release. This significant advance can enable key increases in both cell specificity and therapeutic efficacy through incorporation of well-defined combinations of targeting ligands as well as siRNAs, ultimately creating hybrid structures that fuse the delivery efficiency of viruses to the design versatility of non-viral vehicles.


Gene silencing therapy based on siRNAs offer unique promise for cancer treatment by providing highly potent and target-specific silencing of genes dysregulated during cancer progression. However, delivery of siRNA remains extremely challenging because of its negative charge and tendency to degrade rapidly under physiological conditions. Furthermore, the unusual genetic and phenotypic signatures in aggressive cancers pose additional barriers for siRNA nanomedicines, as the invasive behaviors in these cells typically originate from simultaneous alterations in multiple cell surface receptors and gene expression profiles. Accordingly, there is an unmet need for therapeutic strategies able to more accurately target these cells by recognition of their specific balance in surface receptor expression, with the subsequent triggered release of appropriate siRNA cocktails. Design of such approaches could provide significant improvements in therapeutic efficacy relevant to metastatic cancers and a variety of other diseases. In this proposal, our objective is to design highly tunable, multi-functional Hepatitis B Virus (HBV) capsids suitable for cell-specific siRNA delivery in inflammatory breast cancer (IBC) cells, a canonical aggressive cancer whose invasive behavior is defined by multifactorial changes in gene expression. 



8. Design of RNA-triggered Disassembly Mechanisms in Multi-responsive Polymer Nanocapsules for Personalized Physiological Profiling and Tailored Therapeutics (NSF)

This research addresses the National Academy of Engineering’s Grand Challenge to engineer better medicines by developing new approaches that will ultimately allow rapid assessment of the genetic profiles in patients and the release of personalized drug cocktails.  Our approach is to incorporate DNA “strand displacement” biosensing designs within polymer nanocarriers such that the DNA interface controls nanocarrier stability.  Recognition of specific RNA or DNA sequences will result in a strand displacement reaction that destabilizes the nanocarrier and allows the release of encapsulated drugs or diagnostics.  The major milestones in this work include the design of DNA strand displacement designs sensitive to breast cancer-specific RNAs; incorporation of these DNA duplexes within block polymer micelles; establishment of the specificity and efficacy of RNA-mediated nanocarrier disassembly; addition of cancer cell-targeting peptides on the surface of the nanocarriers; and establishment of selective cellular uptake and efficient cargo (dye) release in breast cancer cells. 




9. Synthetic Control of Metabolism by Dynamic Metabolons (NSF)

The ability to optimize pathway flux is one of the most important factors toward maximizing product titers. The traditional approaches largely focused on the overexpression of rate-limiting enzymes, competing pathway deletion, and resource management. However, most of these approaches are static in nature and do not provide dynamic regulation of pathway fluxes based on substrate, precursor, and product availability. Although many genetic circuit designs have been implemented to provide dynamic control of gene expression and pathway fluxes, these dynamic strategies usually provide only excellent “up-regulation” control but down regulation is much slower as it requires the degradation of the associated regulatory components. In this proposal, we seek to investigate a new transformative strategies that provide faster on-and-off control based on enzyme proximity control inspired by natural dynamic metabolons. The goal to develop synthetic dynamic metabolons that will allow carbon flux to be redirected at will. The end result is the ability to provide dynamic optimization of microbial metabolism for optimal product formation. This will be achieved by the dynamic shifts between the assembly and disassembly of synthetic metabolons in order modulate the overall output function. Nature has already based on the use of RNA scaffold. The central framework is to design RNA-based dynamic metabolons that assemble in response to specific metabolic demands and to exploit the dynamic shift between the assembly and disassembly of enzyme complexes to coordinate metabolic pathways for optimal product titer. The tools developed here could be transferred to other organisms, and used to address fundamental questions about control and regulation of metabolism.


10. Repurposing the CRISPR-Cas9 system for dynamic control of cellular metabolism (NSF)

Cellular metabolism is capable of highly specific and efficient chemical synthesis at mild temperatures and pressures far beyond the capability of most synthetic chemical routes. Although pathway engineering can be used to further improve the range of compounds that could be synthesized, achieving commercially viable productivity remains challenging. An emerging strategy to combat these issues is to organize pathway enzymes into a sequential multi-enzyme complex in order to improve the overall pathway flux, to minimize cross-reactions, and to provide kinetic driving forces that redirect the carbon flux through essentially reversible steps. Although synthetic biologists have taken a more modular approach using biomolecular scaffolds to co-localize target enzymes, the current approaches either lack the required binding affinity or flexibility in tuning the cascade assembly. To address these shortcomings while providing a highly modular and flexible platform for assembling in vivo enzyme cascades, we propose here a new and potent approach that enables specific and high-affinity binding to DNA scaffolds using a special version of the CRISPR/Cas9 system. By combining the ability to provide site-specific, dCas9-guided enzyme assembly and the ability to provide disassembly by controlled dCas9 degradation, the overall objective of the proposal is to develop a transformative framework to create highly efficient and dynamic enzyme cascades suitable for many synthetic-biology and metabolic engineering applications.


While the native function of Cas9 is for RNA-guided DNA cleavage, nuclease-null Cas9 (dCas9) proteins have been generated while preserving the same high-affinity DNA binding capability. Our main objective in this proposal is to repurpose the dCas9 system as a generalizable platform for site-specific enzyme assembly and to demonstrate its utility to assemble enzyme cascades onto DNA scaffolds for metabolic engineering and synthetic biology applications. Unlike other dsDNA targeting platforms for which custom ZFPs and transcription-activator-like effector (TALE) proteins remain somewhat difficult and expensive to design for each unique target sequence, the dCas9-based approach offers the ease of tuning the sequence specificity simply by using a new sgRNA. A set of orthogonal dCas9 proteins will be repurposed as a new tool for in vivo assembly of enzyme cascades. The ability to provide both subnanomolar binding affinity and site-specific localization onto a simple plasmid DNA template will revolutionize our ability to create a diverse class of synthetic enzyme cascades for optimal pathway engineering applications. By combining metabolite-responsive dCas9 expression and controlled dCas9 degradation to provide both fast sensing and actuating ability into an integrated synthetic design, we believe this new framework will lay the foundation as a new transformative approach for implementing dynamic control of cellular metabolism.



11. Advanced Biomanufacturing of Functional Bionanoparticles for Bioimaging (NSF)

Research on nanomaterials in the past decades has undergone explosive growth. Although recent progress in nanomanufacturing technologies led to successfully large-scale manufacturing of a range of inorganic and organic nanostructured materials, the extension of those manufacturing processes to the production of advanced functional bio-nanoparticles (bio-NPs) is currently an unmet challenge. This is mainly due to the sensitivity of bio-NPs to the harsh conditions of current manufacturing processes. The goal of this proposal is to design an advanced biomanufacturing process to manufacture genetically engineered multi-functional bio-NPs. The local and global structure of the advanced bio-NPs will be monitored and characterized, and their functionality and utility will be examined and validated in bioimaging of brain tumor in mouse model.


Our hypothesis is that by employing genetically engineered vesicle-forming bacteria as microbial cell factories, we will reliably produce uniform bio-NPs with precisely controlled biological functionality in a continuous and scalable way. To accomplish it, recombinant DNA technology will first be used to design novel genetically engineered protein multi-functional bio-NPs for capture and detection functions in bioimaging. The bio-NPs are lipid-based outer membrane vesicles (OMVs) with a uniform diameter of ~50 nm and the outer leaflet of the bilayer is decorated with novel engineered protein fusion, endowing multi-functionality. The OMVs, co-displaying multiple copies (~50, each) of super-active NanoLuc luciferase enzyme (~150-fold more active than other luciferases), will contain (i) an antibody-binding domain (ZZ domain) for anchoring antibodies of interest, and (ii) a thermo-responsive elastin-like protein domain (ELP, soluble at room temperature and insoluble/aggregated at 42 °C) for simple purification of the OMVs via size filtration. A fermentation process integrated with two-stage size filtration will then be designed for continuous, sustainable production of multi-functional OMVs at a large quantity.