Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Isaacs, Farren

Abstract

Plants have evolved to produce a myriad of secondary metabolites, which have been a rich source of small molecules with numerous applications in modern medicine. However, many therapeutic small molecules still rely on plant sources for their production and confront various challenges such as low purification efficiency, slow growth of plants, and the scarcity of species. Advances in synthetic biology have created alternative opportunities to overcome these challenges by producing valuable plant natural products through heterologous expression of the biosynthetic pathways in microorganisms. However, the biosynthetic pathways of many plant natural products remain poorly understood due to inaccurate gene annotations and time- and cost-intensive experimental validation of large numbers of putative enzymes.Camptothecin (CPT), a plant-derived monoterpene indole alkaloid first identified in Camptotheca acuminata, is a drug precursor widely used for cancer chemotherapeutics. However, the full set of genes responsible for CPT biosynthesis remains unclear, hindering efforts to elucidate the complete pathway or establish biosynthetic production of CPT in heterologous hosts. In this thesis, experimental, computational and synthetic biology technologies was employed to provide new insights into CPT biosynthesis and heterologous expression of the biosynthetic pathway. In Chapter 2, I identified spontaneous formation of key CPT precursors, providing an additional path to CPT biosynthesis. To advance our understanding of the genes involved in CPT biosynthesis, I also engineered an experimental callus system for inducible production of CPT, which enabled multi-omics and deep learning analyses. In Chapter 3, an improved genome assembly and gene annotation for C. acuminata was generated to lay the foundation of gene identification. I then leveraged the natural variation of CPT levels in C. acuminata tissues and performed transcriptomic analysis of multiple callus and tissue types to shortlist candidate enzymes responsible for CPT biosynthesis. Finally, large-scale deep learning-enabled protein-ligand complex structure prediction was conducted to prioritize 117 candidate enzymes for experimental validation in bacterial and yeast expression systems. In Chapter 4, functional validation of the shortlisted candidate genes was carried out in both Escherichia coli and Saccharomyces cerevisiae using in vivo or in vitro functional assay. However, technical challenges in heterologous expression of many candidate enzymes confounded data interpretation, leading to inconclusive results for the identified candidate list and warrants further investigation in future studies. In Chapter 5, a computational and experimental synthetic biology technology was developed to maximize the discovery of secondary metabolism, which enables broad-host-range expression of biosynthetic pathways of natural products in Gram-negative and Gram-positive bacteria as well as eukaryotes using conjugation and landing‑pad strategies. I also found a single mutation in the RSF1010 origin that permits replication in Bacillus subtilis. By integrating experimental, genomic, transcriptomic and deep learning approaches, this thesis provides a valuable foundation for the elucidation of CPT biosynthetic pathway, paving the way for sustainable production of CPT and its derivatives in heterologous hosts. A broad-host-range expression system was also developed to enable decoupling of biosynthetic capacity from host‑specific regulation and metabolic context, effectively enabling expression of otherwise silent or inaccessible pathways across multiple hosts.

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