1. Retrobiosynthesis
Previous publications: Nature Catalysis 2025
We recently completed the digitized recording of more than 100,000 reactions from thousands of modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs), and established the ClusterCAD database along with the open-source automated design tool ClusterCAD RetroTide. This tool can automatically design and optimize the selection of PKS/NRPS modules based on the target product structure. To practice ClusterCAD RetroTide, I first used simple but industrially important targets (diols and amino alcohols) that require only two PKS modules for synthesis. The simpler synthesis allowed me to focus on establishing chassis strains suitable for practicing PKS engineering. Using integrative technologies in metabolic engineering, enzyme mining, structural biology and automated computational design, I integrated versatile PKS initiation and reductive termination strategies into designed pathways. As a result, 17 industrial diols and derivatives were biosynthesized in harnessed Streptomyces chassis at high titers, demonstrating the feasibility of designing such a biomanufacturing platform.
Our future goal is to systematically overcome the challenges of PKS/NRPS engineering to complete “a thousand molecule project”. With our mature digital design tools and harnessed hosts, I plan to achieve the rapid and efficient assembly of 5–10 PKS/NRPS modules and biosynthesize over 1,000 new-to-nature small molecules in the short term and 10,000 molecules in the long term.
2. Enzymology
Previous publications: Nature Chemistry 2019
Prenylated fungal indole alkaloids with a bicyclo[2.2.2]diazaoctane core are of significant interest due to their broad biological activities, including calmodulin-inhibitory malbrancheamides and anthelmintic paraherquamides. We elucidated the malbrancheamide biosynthetic pathway using biomimetic total syntheses to generate racemic alkaloids and intermediates, along with in vitro enzymatic reconstitution to yield the natural antipode (+)-malbrancheamide. Our work revealed a common NRPS biogenetic scheme involving dipeptide reductive cleavage and a cascade of reactions leading to intramolecular [4 + 2] hetero Diels-Alder (IMDA) cyclization. The bifunctional enzyme MalC, a reductase/Diels-Alderase, is crucial for synthesizing optically pure (+)-malbrancheamide with strict stereocontrol, and we determined the structural basis for IMDA catalysis. Taken together, these new discoveries provide a novel enzymatic toolkit for developing improved anthelmintic therapeutics to address human and animal diseases.
Nature Communications (Accepted)
Reducing carbon emissions from aviation and long-distance transportation sectors requires the development of sustainable biofuels with suitable energy density, freezing point, and other physical properties. We previously demonstrated biological production of high energy polycyclopropanated fatty acids (POP-FAs, class I) using an iterative polyketide synthase (iPKS) pathway in a Streptomyces host. We next explored the natural diversity of POP biosynthesis by investigating homologous pathways. By in vivo gene exchange, we determined cyclopropanase (CP) catalysis to be key for POP-FA engineering. Leveraging both natural and engineered pathway product diversity, we demonstrate targeted production of new classes of POP-FAs, namely shortened POP-FAs (class II) with distinct cyclopropane positions, as well as fully cyclopropane-saturated POP-FAs (class III). Compared to class I POP-FAs, shortened class II POP-FAs are predicted to have superior freezing point properties for aviation, while class III POP-FAs should have superior energy-density. These precise and controllable modifications to POP-FA structure open the door for bioproduction of designer POP fuels. More importantly, further dissecting CP catalysis will guide engineering the biosynthesis of POP molecules that are very challenging to synthesize chemically.
In collaboration, we have combined the ClusterCAD RetroTide tool with AI-guided design for post-PKS tailoring enzymes. This BioPKS Pipeline integrates the logic of designing PKSs for carbon backbone assembly and mining tailoring enzymes for functional group diversification, greatly expanding the range of compounds accessible through computational design. To demonstrate its application potential, we identified a new P450 ketone synthase and built unnatural PKS-P450 pathways for rapid bioproduction of industrial ketones. In the future, I plan to enable many major types of enzyme decoration onto common PKS/NRPS core structures, featuring programmable access to precisely tuned chemistry. Using the ESM language model, which captures sequence, structural, and functional aspects of protein evolution, we will also develop low-N evolution strategies that only require a small number (“N”) of experimentally tested variants to achieve improved or even new protein functions.
3. Metabolic Engineering
Previous publications: Coming soon
Over the past few decades, bioorthogonal chemistry, which allows chemistry to be installed in microbes without disrupting native biochemistry/metabolism, has transformed the interface between chemistry and biology. In parallel, advances in directed evolution and genome mining have vastly expanded chemical reaction sets available within living systems. The full integration of these once “abiotic” reactions into cellular metabolism has now become a rapidly emerging field, and it raises a fundamental question: how do microbes respond to the new reactions we engineer them to perform? The accumulated knowledge will contribute to the development of improved microbial hosts.
We began to explore this question using our diol biosynthetic pipelines, where designed unnatural PKSs produce large quantities of aldehyde intermediates that are highly toxic to host cells. This stress resulted in the accumulation of hundreds of genome-wide mutations. Through detailed investigations, we engineered detoxification pathways to mitigate the cellular burden; as a result, we largely lowered mutation rates and achieved more than ten-fold increase in diol titers.