Plant Synthetic Biology

Background Introduction

Nowadays, there is a rising global focus on human health issues. Many countries are intensifying anti-smoking efforts through measures such as prohibiting the sale of tobacco to minors and increasing tobacco market taxes. However, as the large scale of the tobacco industry which produces high economic value and employs the significant number of people, serves as an crucial source of tax revenue for some countries, the trend of smoking ban will inevitably limit the profits and employment rates generated by the tobacco industry, which may impact national development. To facilitate smoother anti-smoking initiatives and minimize associated interest losses, the tobacco industry is faced with the challenges of de-stigmatization and industry transformation and upgrading.

N.benthamiana is a native herbaceous plant in Australia, characterized by its dicotyledonous secondary growth properties. It is an important model organism for botanical research and serves as a crucial plant platform for the production of biopharmaceutical proteins and vaccines. It plays a critical role in studies related to synthetic biology, metabolic pathway engineering, functional genomics, and gene editing.

Considering the merits of Nicotiana benthamiana over other plants, such as low cultivation costs, short growth cycles, high genetic transformation and transient expression efficiency, and the convenience of purifying recombinant proteins, we believe that promoting the application of N.benthamiana and other tobacco plants as chassis for producing natural medicines in the biomedicine sector aligns with the domestic and international market demand for large-scale production of rare natural medicines. This approach can effectively address potential issues facing the tobacco industry in the future.

Problem Statement

In preliminary research, this project discovered that the level of chlorogenic acid in natural Nicotiana benthamiana is excessively high. This results in a significant diversion of the metabolic flow necessary for producing valuable secondary metabolites, thereby greatly limiting synthetic efficiency. We aim to utilize synthetic biology techniques to modify the metabolic flow of natural Nicotiana benthamiana, creating an improved chassis to enhance its production capacity and provide a more diverse range of plant chassis options for future pharmaceutical production and other fields.

Solution to the Problem

To address the aforementioned issues and achieve the project goals, we constructed a genomic-scale metabolic network model (GSMM) for Nicotiana benthamiana and used the COBRA algorithm to analyze and devise optimal metabolic flow allocation strategies. Guided by the modeling results, we selected different combinations to knockout the HQTs gene in the Nicotiana benthamiana genome, successfully developing a chassis with low chlorogenic acid levels.

To validate the chassis's potential for synthesizing different types of high-value compounds, we introduced three heterologous biosynthetic pathways by selecting enzyme genes optimized for codon use in Nicotiana benthamiana. These pathways synthesized caffeoylmalic acid, resveratrol, and crocin, aiming to verify the synthesis potential of this new low-chlorogenic acid plant chassis in the upstream and downstream of the shikimic acid pathway, as well as in related bypass MEP metabolic pathways. Ultimately, we successfully demonstrated that under low chlorogenic acid levels, the metabolic flow in Nicotiana benthamiana shifted more towards the synthesis of high-value compounds, indicating that the improved chassis has a much higher synthetic potential compared to traditional Nicotiana benthamiana.

Moreover, to further enhance product yield, we employed semi-rational enzyme design and directed evolution methods to modify and improve the catalytic activity and thermal stability of key enzymes involved in the introduced foreign metabolic reactions, with the aim of increasing product value. Experimental results indicated that the modifications made to key enzymes in the metabolic pathway increased the yields of certain target products to some extent.

Overall, our project successfully constructed a improved Nicotiana benthamiana chassis with low chlorogenic acid levels. Through well-designed dry and wet experimental approaches, we effectively demonstrated the efficacy of this new plant chassis in addressing the plant synthetic biology issues raised in this project. The new plant chassis developed in this project received valuable feedback from presentations and discussions with other teams, which helped us advance our project. Moreover, It has been highly recognized by different teams for its practicality and other aspects. We hope that in the future, the new plant chassis we create can assist other teams in better addressing plant synthesis-related challenges or inspire other teams to improve certain model plant chassis.