Project background

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Cancer is one of the most serious health issues facing human society. As one of the primary treatment methods, chemotherapy currently faces significant challenges, including severe side effects and limited efficacy. An isothiocyanate compounds found in cruciferous vegetables, particularly in broccoli seeds, sulforaphane, have been found to exhibit excellent anticancer properties with minimal side effects.

However, its production relies on extraction from broccoli seeds, which contain less than 2% sulforaphane. This extraction process requires extensive broccoli cultivation and is affected by the plant's growth cycle, leading to land wastage, high costs, and unsustainable production issues.

Our goal is to biosynthesize sulforaphane from scratch using Saccharomyces cerevisiae and optimize its yield through computational and synthetic biology techniques. By reducing costs ^🔗, we can minimize land waste and deforestation, promote human health, and provide a sustainable alternative aligned with global climate change mitigation efforts.

Lab

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Given that sulforaphane synthesis relies on various plant-derived enzymes, we chose Saccharomyces cerevisiae as the eukaryotic chassis. Our goal is to construct a de novo biosynthetic pathway for sulforaphane in yeast. We have obtained permission from the safety committee to use Saccharomyces cerevisiae.

This process involves a series of complex reactions, and we aim to maximize yield while constructing the entire pathway. We plan to optimize and modify members of the Arabidopsis P450 enzyme family, as existing research indicates potential for further optimization. Given the high conservation of the P450 family, our strategy includes computational enzyme engineering, protein optimization, and the development of more efficient “enzyme factories” to achieve higher yields.

Metabolic Simulation

To identify the optimal pathway for sulforaphane biosynthesis in yeast, we aim to use computational techniques to develop algorithms that simplify the yeast metabolic network, optimize metabolic flux, and simulate and screen various pathways for sulforaphane production. Through screening and comparison, we will enhance the yield of existing pathways or optimize them.

Construction of the De Novo Sulforaphane Biosynthetic Pathway in Yeast

Based on transcriptome analysis, researchers identified the natural metabolic pathway for sulforaphane synthesis. Building on this, and through metabolic simulation and optimization, we have constructed a de novo sulforaphane biosynthetic pathway in Saccharomyces cerevisiae.

Engineering of P450 Enzymes

The cytochrome P450 family often exhibits inefficiency during heterologous expression due to its high conservation and complex functional systems. To address this challenge, we plan to use computational design to optimize two Arabidopsis P450 enzymes in the pathway. We will enhance their catalytic efficiency by adjusting the hydrophobicity of their substrate channels, modifying their subcellular localization, and increasing cofactor supply. We hope these multifaceted strategies will further optimize sulforaphane yield.