Module1

Further increase the yield of borneol

In our work, synthetic pathway has been optimized as much as possible, so we will mainly focus on upstream and downstream of the pathway in the future.

Seeing acquisition of the substrate as the upstream part of the MVA pathway, it’s rewarding for us to further increase its efficiency. When we use fat as the carbon source of Y.lipolytica, the absorption efficiency depends on the efficiency of decomposing it into fatty acids. We plan to increase the expression of extracellular lipase Lip2 to make Y.lipolytica obtain more fatty acids. Then in order to efficiently transport so many fatty acids into peroxisomes, we will highly express YlFaa1p protein in Y.lipolytica to activate fatty acids in the cytoplasm, and increase the expression of ABC transporters YlPxa1p and YlPxa2p to transport activated fatty acids into peroxisomes (Figure 1).

Figure 1. A schematic diagram of the way Lip2, YlFaa1p and YlPxa1/2p work.

β-oxidation can provide a large amount of raw material acetyl-CoA for the synthesis of borneol, but its efficiency is limited by several key enzymes. We hope to increase the expression of rate-limiting enzymes, such as POT1, to facilitate the reaction. Additionally, less ATP in the peroxisomes may hinder the reaction. In order to change this situation, we will express an ATP transporter, ANT1, on peroxisomes, to increase ATP supply in this organelle (Figure 2).

After the modification above, our strain will produce much more borneol. However, the reaction will be hindered if the product accumulates too much in the peroxisome. What’s more, the accumulation of terpenoids will be harmful to cell. Although the oil droplets in Y.lipolytica can store borneol, we still hope to transport excessive borneol out of cells. To this end, we will express the terpenoid transporters LaABC-B1 and GcABC-G1 on both peroxisomes and cytomembrane of our strain (Figure 2).

Figure 2. A schematic diagram of the way YlAnt1p, LaABC-B1 and GcABC-G1 work.

In addition, because our strain has the ability to produce a variety of terpenoids, we hope to use this strain to efficiently produce other substances in the future, such as limonene, lycopene, etc. We also hope to simultaneously produce liposome encapsulated drugs and cross-linked drugs, such as patchouli alcohol - borneol co-production.

Module2

1. Optimize the interaction between the biosynthesis of borneol and the produce of Bor-Nps.

As the design showed ,we have difficulties in converting borneol into the corresponding ketones. We have to use chemical process to achieve this conversion. But we wish to finish all the process in the biological systems,but there is no such an enzyme to finish this work. So we seek help from our partnership ,USTC-Software. They helped us find many enzymes which may possess the catalyze ability with their software. And we are going to conduct experiments on these enzymes and test their ability to produce the corresponding ketones.

Figure 3.

2. Optimize the targeting ability to the blood–brain barrier

Our Bor-Nps doesn't have a specific binding site with the cells of the blood-brain barrier now. So our Bor-Nps may fuse with any cells in the human body. It will lower the efficacy of the drug we are delivering. So we are going conjugate some molecule to help our Bor-Nps to target the blood-brain barrier. There are some special receptors on BBB endothelial cells, such as transferrin receptor, lactoferrin receptor, epidermal growth factor receptor, N-acetylcholine receptor, amino acid transporter, glutathione transporter and glucose transporter.

Glucose transporter type 1 (GLUT1) is highly expressed on the cell membrane of brain capillary endothelial cells, and the transport efficiency is the highest. The glucose consumption of the human central system is equivalent to 30% of the total body consumption, and the glucose transported to the brain by the brain endothelial cells is tens of times its own weight every minute. Therefore, the drug molecules modified by glucose should also have good brain targeting. So we assume that the nanoparticles loaded with the glucose residue would help a lot to improve the targeting ability.

3. Bor-Nps effectiveness test

We will perform animal experiment to test if the Bor -Nps really makes outstanding contribution in delivering drugs. We will first test the toxicity with mouse through Bor-Nps intravenous injection and observe the vitality of the mouse. Then we are going to load hydrophilic drugs with our Bor-Nps and intravenous inject them into the mouse,and then detect the content of the drug in the mouse brain compare with the normal nanoparticles drug delivery system. We wish we will learn the effectiveness of the Bor-Nps we have built.

Module3

Future-Mass Production in Factory

In future, we also plan to introduce our project to actual production. As the flow chart shown below, we have designed the whole process of factory production.

In our previous experiment, we determined the optimum culture conditions at relatively small scale in the laboratory. For mass production, we’ll take the size of fermentation tank, the dissolved oxygen level and the content of nutrients into consideration.

Besides, to guarantee the security, we have designed a suicide gene circuit in our strains which can be activated by blue light and Cu²⁺ in the solution. A sensor inside can monitor the temperature, humidity and absorbance, which could be used to analyze the growth of yeast and determine when to send the fermented broth to next procedure. At the end of fermentation, the light generator will be turned on meanwhile the Cu²⁺ will be released into the solution. Then the light-inducible transcription factor and the ion-sensitive promoter will initiate the transcription of Cas9, which can efficiently knock out RAS1/2 under the guidance of sgRNA. Noticeably, the system will not be able to work if there is only one activator (blue light or Cu²⁺), which can reduce the risk of start by mistake and avoid loss caused by it.

In the purification, we will conduct a series of experiments to determine the appropriate speed to isolate the LNPs with other components. Given that some of borneol may remain in the aqueous phase, a step of extraction can be taken if necessary.

To protect the environment, the waste in the ferments will not be simply thrown out. Instead, it will be sterilized carefully and transferred into feed. In order to apply this environmental friendly strategy to actual manufactory, we’ll devote ourselves to improve our recycle system and meeting the standard of food safety.

ZJU-China

Team:USTC