Our project is based on the fermentation industry. We are looking forward to using our projects in the industry. Our target users are fine chemical synthesis companies. To implement our project in the real world, we have to proves that our project is safe and efficient.
Safety
The chassis strains used in our project are E. coli and Saccharomyces cerevisiae, which are widely used in industrial production and be very safe. We strictly control the biosafety during the experiment to ensure that the devices, methods, and materials we use are very safe and do not cause any harm to the human body or the environment.
Escherichia coli and Saccharomyces cerevisiae, the most commonly used microorganism in industry, were selected as co-cultured strains. They will not proliferate if they leave the fermentor.
So we decided not to use suicide genes to control them.
Non-toxic
The products, intermediates, and mediums are non-toxic.
Through our biosynthesis progress, tyrosine is the precursor in the Eriodictyol metabolic pathway, which is non-toxic. P-coumaric acid as an intermediate product is the result of many considerations, including its non-toxic advantages. While the terminal product Eriodictyol as natural editable flavonoids has been proved to be safe with no doubt. Meanwhile, we carefully ensured that the enzyme we used wouldn't do any harm to human beings or the environment.
Strains
In our expectation, the growth rate of S.cerevisiae will be an important factor restricting the synthesis rate of products. We will choose aciduric E. coli to adapt to acidic culture medium which is beneficial to S.cerevisiae growth.
Enzyme choose
All of the enzymes are from the literature and iGEM database, we chose the ones that have the highest enzyme activity.
All of the enzymes are codon-optimized to improve expression efficiency.
Take CHS （chalcone synthase）as an example. This enzyme has got various most suitable PH when using different substrates. Besides, its different classifications react differently to the accumulation of flavonoids. We've considered multiple factors in order to maximize the ultimate production. Other enzymes have been selected based on plenty of investment as well.
Model results
Before the experiment, we used the model in the papers to carry out a simple simulation, and the results were satisfactory. For some special reasons, we only carried out experiments on cell growth under co-culture conditions, and we modified the model based on the data obtained. We verified the modified model again and compared it with other co-culture models, and found that our model was more efficient and in line with our expectations. In the study of flavonoid production pathway, we found that co-culture is the most efficient way to produce flavonoids. Therefore, on the whole, our model is efficient and meaningful.