(2ek) Discovery and Engineering of Ribosomal Peptide Natural Products for Therapeutics

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) constitute a major family of natural products that have been found in all three domains of life. To date, RiPPs have been reported with vast structural diversity and hence a broad range of biological functions, including antimicrobial, anticancer, and antiviral activities, making them an ideal source for drug development. The explosion of genomic data and the advancement of genome mining tools have enabled the identification of RiPP biosynthetic gene clusters (BGCs) in silico, however, attempts to isolate these compounds often fail due to the negative regulation over these BGCs under standard laboratory conditions, a common problem for traditional natural product discovery. To rapidly explore their biosynthetic potential in nature, my research focuses on the genome mining of RiPPs by leveraging synthetic biology approaches. Candidate RiPP BGCs predicted by bioinformatics are categorized into two groups: the high-fidelity group and the class-defining group. The high-fidelity group comprises BGCs that are annotated as characterized RiPP classes yet highly underexplored. Given the high successful rate of refactored BGCs in heterologous expression, an automation friendly plug-and-play method was developed to refactor the high-fidelity BGCs efficiently. While BGCs in the class-defining group encode unknown and usually complicated biosynthetic machinery, a direct cloning strategy named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) is used to manipulate these BGCs for heterologous expression. Afterwards, the initial product screening is performed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Due to the high efficiency and low time consumption of these synthetic biology approaches, the genome mining of RiPP BGCs can be achieved rapidly. The effectiveness of pathway refactoring in uncovering high-fidelity RiPPs was demonstrated by uncovering a class iv lanthipeptide and four glycocins with interesting structural features and bioactivities, which significantly expanded these two previously underexplored RiPP classes. In addition, two novel classes of RiPPs, named daptide and lipoavitide, were also discovered by the direct cloning approach and characterized with unprecedented biosynthetic machineries. Overall, the synthetic biology based approaches were demonstrated to be highly useful for unveiling novel RiPPs, and have the potential to be developed into a scalable platform for RiPP genome mining.

Research Interests

Built upon my knowledge background on synthetic biology and chemical biology, as well as my research experience on RiPP discovery and characterization, my future research interest will focus on the discovery and engineering of peptide natural products, as well as the development of therapeutic peptide-producing probiotics, to promote human health. Specifically, my research interest can be divided into three aims: 1) profiling RiPPs encoded in the metagenomics data by hybrid computational and synthetic biology approaches; 2) cell-free synthesis and engineering of macrocyclic peptides with pharmaceutical potentials; and 3) development of therapeutic peptide-producing commensal bacteria as next generation probiotics.

Profiling RiPPs encoded in the metagenomics data by hybrid computational and synthetic biology approaches.

RiPPs are characterized with high structural diversity and broad range of biological functions, revealing its invaluable therapeutic potential. These peptide natural products are widely encoded in commensal microbes of human beings and have critical roles in mediating microbe-microbe and microbe-host interactions. Discovery and characterization of these microbiome-derived RiPPs not only provide prominent drug candidates, but also render a unique angle for ecological understanding and engineering of microbiomes that are vastly important in human health. However, our current understanding of these microbiome-derived RiPPs is largely restricted by their accessibility. Genome mining of RiPPs from microbiome samples is hindered in multiple facets which includes, but not limited to, the unculturable nature of commensal microbes, trace or no expression of the biosynthetic pathways of interest, and challenging de novo genome assembly of potential producers. Given the considerable progress in mechanistic understanding of RiPP biosynthesis, together with the increasing confidence in predicting RiPP precursor peptides through bioinformatics, I’m interested in leveraging computational and synthetic biology approaches to rapidly profile the RiPP biosynthetic potential encoded in the metagenomics data, characterize their biosynthetic routes, and investigate their biological and ecological functions.

Cell-free synthesis and engineering of macrocyclic peptides with pharmaceutical potentials

Macrocyclic peptides isolated as natural products are of vast pharmaceutical importance due to their desirable biological activity and in vivo stability. Naturally synthesized as nonribosomal peptides (NRPs) and RiPPs, macrocyclic peptides usually contain diverse nonproteinogenic amino acid residues and ring connectivities. The structural complexity are critical for their biological functions, but also inevitably makes the synthesis and engineering of macrocyclic peptides very challenging. By leveraging the cutting-edge technology of cell-free protein expression, and understanding of enzymatic macrocyclization in RiPP biosynthesis, I’m interested in developing a pipeline for synthesizing macrocyclic peptides with designated amino acid residues and ring connectivities, followed by mRNA display-based high-throughput screening for target-binding peptides with increased affinity.

Development of therapeutic peptide-producing commensal bacteria as next generation probiotics

Probiotics are living microorganisms that confer beneficial effects on human health. They act via interaction with both the host and host-associated microbiota and target a variety of sites including the gut, oral cavity, vaginal tract and skin. Despite the range of organisms with potential health benefits has dramatically expanded, the majority of the probiotics are from a limited list of genera, which largely restricts the potential application of probiotics. My research will focus on developing human commensal non-model bacteria as the next-generation probiotics. I’m particularly interested in developing genetic tools to harness the metabolic capacity and niche selectivity of representative species, and engineering them as precise delivers of peptide therapeutics to different sites of the body.

Teaching Interests

As Albert Einstein’s famous quote saying “education is what remains after one has forgotten what one has learned in school”, I always believe that the ultimate goal of education is to train students with the capacity to independently acquire knowledge of what they need, particularly in this information explosion era. Therefore, my teaching philosophy centers on critical thinking and practice, which I found by my personal experience the best way of training an independent learner. To achieve this goal, active learning principles will be extensively applied in my course design. Stimulation of curiosity would always be the main rhythm of my classes. Students are encouraged to learn broadly and think thoroughly. Students will also be presented with plenty of opportunities to engage into team projects, which is an effective way to encourage them to learn collaboratively.

I have been a teaching assistance for three semesters during my doctoral training and involved in fundamental chemical engineering laboratory and process engineering courses, which I found as an invaluable experience to learn instruction, course and exam design and communication with students. My future teaching interest includes undergraduate level fundamental chemical engineering courses such as transport phenomena and thermodynamics. Regarding to the graduate level courses, I would like to focus on classes related to synthetic biology and chemical biology, as I can share my research experience and vision as an active scientist in this field with graduate students who are more eager for these information.