Team:USTC
Background
The brain is the core of our body with complex functions and unique architecture. In order to transfer accurate information and regulate the body functions, the neuronal cells require a critically maintained microenvironment.
The blood-brain barrier (BBB) is a physical and metabolic barrier which provides the key mechanism for maintaining the appropriate stability for neuronal function. The inner layer of BBB is mainly composed of endothelial cells on the brain capillary walls and tight junctions (TJs), which hinder the transportation of polar molecules and macromolecules through the paracellular between the adjacent endothelial cells.
But it can be a double-edged sword while the brain encounters meningitis, epilepsy and other neurological diseases, because many active components of drug cannot be efficiently delivered to the site of action. According to the FDA (Food and Drug Administration), most of the brain-targeted drug candidates have been withdrawn for the absence of capability of crossing the BBB. Hence, the efficiency and accuracy of drug delivery becomes the key problem of current therapy of cerebral disorders.
To tackle with this problem, we, the USTC iGEMers, decided to find a solution from traditional Chinese medicine. And we learned that camphor is an effective drug in the therapy of epilepsy from the ancient classic Compendium of Materia Medica by pharmacist Shizhen Li. And the main component of camphor—borneol plays an important role in increasing the permeability of BBB, which means it can be a good assistance to drug delivery in human brain. Since thousands of years ago, our ancestors have used borneol as a powerful drug in treatment of epilepsy and meningitis and created traditional prescription of bezoar bolus for resurrection.
For its superior performance in crossing the BBB, borneol is widely used in the surface modification of LNPs (lipid nanoparticles) to load brain-targeted drug. However, due to possible toxic side reactions (e.g. isoborneol) and the relatively low chiral selectivity, chemically synthesized borneol cannot meet the standard of purity and safety. Today’s borneol for medical use still highly depend on extraction from dipterocarp（Dipterocarpus turbinatus Gaertn.f.）. But the efficiency of extraction from dipterocarp is very low. According to the data from Chinese natural borneol industrial report in 2018, every acre of dipterocarp could only produce 3kg borneol. What’s worse, the resource of dipterocarp is at the edge of depletion because of deforestation. On the contrary, the huge demand of borneol with high purity is still increasing today, which makes it too expensive to use in mass production.
On the other hand, the number of patients with neurological diseases is still increasing. It is estimated that in China every year there are 40,000 new cases of epilepsy. Those diseases not only make patients themselves in pain but also brings a heavy burden on their family. With more and more people suffering from different neurological disorders, it’s urgent to develop a new method to manufacture borneol modified drug with both high efficiency and selectivity in large scale.
Luckily, we have the genetic engineering to efficiently biosynthesize our product from the metabolite of microorganism, which inspire us to design a new mode for factory production.
MVA pathway is an essential metabolic pathway widely present in eukaryotes, which transfers acetyl-CoA into isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosohate (DMAPP). DMAPP, the end-product of MVA-pathway, is precursor of most isoprenoids in organisms. In previous study, researchers identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera, which acts as the key enzyme in borneol biosynthesis by converting GPP to bornyl diphosphate. Then they introduced it into GPP high-yield Saccharomyces cerevisiae and obtained the yield of 1.24mg/L.
But we still want to make progress on the current yield. The efficiency of MVA pathway is determined by the supply of acetyl-CoA and activity of enzymes during this process. To solve this problem, we chose a kind of oleaginous yeast Yarrowia lipolytica as our chassis for its native MVA pathway and sufficient supply of acetyl-CoA, ATP and other necessary substrates for terpene synthesis.
Project Goals
We intend to develop a new method to efficiently biosynthesize and extract borneol from Yarrowia lipolytica, then cross-link it with LNPs to produce the borneol modified vehicle for various brain-targeted drug. We also conceive the design of the whole procedure in the factory, which can decrease the cost of drug production.
References
[1] Soni, V., Pandey, V., Asati, S., Jain, P. & Tekade, R. K. in Basic Fundamentals of Drug Delivery (ed Rakesh K. Tekade) 539-593 (Academic Press, 2019).
[2] Xie, J., Shen, Z., Anraku, Y., Kataoka, K. & Chen, X. Nanomaterial-based blood-brain-barrier (BBB) crossing strategies. Biomaterials 224, 119491, doi:https://doi.org/10.1016/j.biomaterials.2019.119491 (2019).
[3] Wang, L. et al. Nose-to-brain delivery of borneol modified tanshinone IIA nanoparticles in prevention of cerebral ischemia/reperfusion injury. Drug Deliv 28, 1363-1375, doi:10.1080/10717544.2021.1943058 (2021).
[4]
Research, L. I. Capacity output and trend development analysis of natural borneol industry in China,
[5] Guo, Q. et al. Harnessing Yarrowia lipolytica Peroxisomes as a Subcellular Factory for α-Humulene Overproduction. Journal of Agricultural and Food Chemistry 69, 13831-13837, doi:10.1021/acs.jafc.1c05897 (2021).
[6] Ma, R. et al. Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae. Synthetic and Systems Biotechnology 7, 490-497, doi:https://doi.org/10.1016/j.synbio.2021.12.004 (2022).
