(4jx) Engineering and Sustainable Production of Advanced Biomaterial

1. Research Interests

Advanced protein biomaterials are versatile for diverse applications due to the programmability of functional protein domains. Alpha helix-based protein material such as macroscopic protein fiber has been overlooked as majority of biomaterial focuses on beta sheet-based protein material inspired by silk. Alpha helix-based protein fibers have remarkable strain (>50%) compared to beta-sheet-based protein fibers (<25%), making them ideal for creating high-toughness material that can absorb impact during the crash and deform without fracturing. My focus lies in investigating alpha-helix based proteins to uncover the principles governing their functionality in material design. Meanwhile, coculture systems have been developed for sustainable production including biomaterial using alternative resources. However, challenges such as growth inhibition between hosts, nutrient competition, and complicated downstream purification persist as roadblocks to sustainable biomanufacturing. My research endeavors are directed towards overcoming these obstacles in sustainable bioproduction. I aim to delve into the underlying mechanisms and employ synthetic biology to develop innovative tools for maximizing the potential of biological systems in advanced biomaterial production.

1.1 Graduate Research (Advisor: Dr. Kevin Solomon, Purdue University)

1.1.1. Characterizing NgAgo and exploring its activities for biotechnological applications

My doctoral training at the Agricultural and Biological Engineering Department focused on developing a novel gene-editing tool utilizing prokaryotic Argonautes (pAgos) sourced from the halophilic Natronobacterium gregoryi (NgAgo). Amidst prior debates on NgAgo's effectiveness in eukaryotic systems, I conducted a comprehensive reassessment of NgAgo both in vitro and within bacterial contexts for its potential in gene-editing applications with advanced synthetic biology tools. Notably, the often-overlooked halophilic characteristics of NgAgo emerged as pivotal factors. Computational analysis unveiled a previously unrecognized single-stranded DNA binding domain, repA, within NgAgo and other halophilic pAgos. This repA domain was then proved to have crucial role. Despite NgAgo's inherent insolubility in low-salt environments, it exhibited DNA cleavage activity upon appropriate solubilization with salt using a cell-free system. I elucidated that this activity, contingent upon both PIWI and repA domains, resulted in either DNA cleavage in vitro or enhancement of homologous recombination and gene-editing capabilities in bacteria (Lee et al., NAR, 2021). Furthermore, I created an optimized selection system aimed at refining endonuclease activity to facilitate future enhancements in pAgo optimization (Lee et al., ACS Synthetic Biology, 2022). Collectively, this work revealed that NgAgo possesses unique catalytic behavior in the pAgo family and has some gene-editing application potential. More importantly, this work expands knowledge of the pAgo family, providing a foundation for future pAgo-based gene-editing tool development.

1.1.2. Engineering virus-like-particles of Barley stripe mosaic virus (BSMV) in heterologous host for diverse applications

Throughout my graduate studies, I undertook the task of engineering the Barley stripe mosaic virus (BSMV) by integrating an RNA loop structure from another host in the transcript of BSMV capsid protein, enabling its benign production in bacteria and coating with metal ions under environmentally friendly conditions (Lee et al., ACS Applied Nanomaterials, 2020). Additionally, I enhanced the structural stability of BSMV's capsid protein to facilitate metal coating under harsh environmental conditions (Vaidya et al., Biochemical Engineering Journal, 2023). This research presents a safe and eco-friendly approach to producing rod-shaped virus-like particles and subsequently coating them with metals for diverse applications.

1.2 Postdoctoral Research (Advisor: Dr. Fuzhong Zhang, Washington University in St. Louis)

1.2.1. Engineering of alpha-helix based biomaterial for diverse applications

The potential of alpha helix-based protein materials has been overshadowed by the predominant focus on beta sheet-based protein materials, often inspired by silk (Jeon et al., ACS Applied Materials & Interfaces, 2023; Lee et al., Molecules, 2023). In this study, I employed an alpha helix protein as a model to investigate its behavior when formed into macroscopic fibers. I characterized its mechanical properties both pre- and post-fiber breakage. By implementing engineering techniques, I enhanced its toughness to levels comparable to Kevlar, a material commonly used in bulletproof vests (in preparation). This research is a foundational step towards the biosynthesis of alpha helix-based materials, offering a roadmap for their application across diverse fields.

1.2.2. Coculturing cyanobacteria and E.coli for sustainable production of advanced biomaterial

Combining cyanobacteria with engineered E. coli has demonstrated the capability to generate valuable compounds. However, the growth of E. coli was notably impeded, constraining its potential for bioproduction. To overcome this obstacle, I investigated the factors limiting bacterial growth and subsequently reinstated the growth of E. coli along with protein production through gene expression modulation and nutrient supplementation (in preparation). This research aims to eliminate the hindrance posed by bacterial growth in coculture scenarios with cyanobacteria, offering valuable insights into developing auxotroph-heterotroph platforms for sustainable biomanufacturing.

1.3 Future Research

In my independent career, I am dedicated to developing novel advanced biomaterials and engineering groundbreaking technologies for sustainable biomanufacturing. My poster will discuss these research directions in more detail.

2. Teaching Interests

Based on my training, I am comfortable teaching General Chemistry, Organic Chemistry, Fermentation Lab, Material Science, and Principle of Molecular Engineering. I can also teach many biology courses aiming for engineering purposes, including Molecular Biology, Genetics, and Biochemistry, as well as interdisciplinary courses that align with my specific research interests in Systems/Synthetic Biology and Biomolecular Engineering.

2.1 Teaching/Mentoring experience

My teaching experiences span various settings, affirming the importance of formal classroom instruction and practical training. In my undergraduate years in Taiwan, I led study groups to assist peers in genetics, molecular biology, and biochemistry. Additionally, I served as a teaching assistant in a molecular biology lab, guiding students in data analysis and drawing conclusions. During my graduate studies at Purdue, I volunteered as a graduate advisor for Purdue's iGEM team, providing mini-lectures and project guidance while designing lab training modules. This contributed to the team's success in winning silver-gold medals.

I have guided over twenty undergraduates and three master's students during my Ph.D. and postdoc. I tailored learning and research goals for each student, aiming to nurture future leaders. Weekly meetings were dedicated to prioritizing research objectives and honing skills to produce publication-quality data. Many of my mentees have pursued graduate studies at esteemed institutions renowned for synthetic biology programs, such as UCLA, Northwestern, and ASU.

2.2 Teaching Philosophy

My academic journey is fueled by a passion for teaching, honed through diverse educational roles. It's fulfilling to equip engineers with lifelong skills, pursued through professional development opportunities where I refined teaching techniques. Notably, modern students possess strong opinions and struggle with distractions like phones. To address this, I have devised strategies to enhance learning efficiency.

To engage students, I employ varied teaching methods, such as problem-based learning (PBL), which is particularly effective in synthetic biology. For instance, I've designed modules where students apply synthetic biology concepts to solve mysteries, enhancing their understanding and retention. Through this process, I instill a deep understanding of the scientific process of obtaining knowledge, aiming to foster independent thinking.

I emphasize applying concepts practically, mirroring real-world challenges. This approach, similar to the iGEM project, empowers students to tackle real-world issues using synthetic biology, preparing them for future careers while highlighting their knowledge's impact.

Selected Publications:

1. KZ Lee, MA Mechikoff, A Kikla, A Liu, P Pandolfi, K Fitzgerald, FS Gimble, KV Solomon. NgAgo possesses guided DNA nicking activity. Nucleic Acids Research (2021) gkab757
2. KZ Lee‡, MA Mechikoff‡, MK Parasa, T Rankin, P Pandolfi, K Fitzgerald, ET Hillman, KV Solomon. Repurposing the Homing Endonuclease I-SceI for Positive Selection and Development of Gene-Editing Technologies. ACS Synthetic Biology (2022) 11, 1, 53–60 ‡ equal contributions
3. YH Lee‡, KZ Lee‡, RG Susler, CA Scott, Longfei Wang, S Loesch-Fries, M Harris, KV Solomon. Bacterial Production of Barley Stripe Mosaic Virus Biotemplates for Palladium Nanoparticle Growth. ACS Applied Nanomaterials. December 2020. ‡ equal contributions
4. AJ Vaidya‡, M Rammohan‡, YH Lee‡, KZ Lee‡, CY Chou, Z Hartley, CA Scott, RG Susler, L Wang, LS Loesch-Fries, MT Harris, KV Solomon. Engineering Alkaline-Stable Barley Stripe Mosaic Virus-Like Particles for Efficient Surface Modification. Biochemical Engineering Journal (2023), 109062 ‡ equal contributions
5. J Jeon, KZ Lee, X Zhang, J Jaeger, E Kim, J Li, L Belaygorod, B Arif, GM Genin, MB Foston, MA Zayed, F Zhang. Genetically Engineered Protein-Based Bioadhesives with Programmable Material Properties. ACS Applied Materials & Interfaces (2023), 15 (49), 56786-56795
6. KZ Lee, J Jeon, B Jiang, SV Subramani, J Li, F Zhang. Protein-Based Hydrogels and Their Biomedical Applications. Molecules (2023), 28(13), 4988