Team:USTC
Module 1 : Efficient microbial production of high-purity borneol in Yarrowia lipolytica
Yarrowia Lipolytica - host for Efficient Synthesis of Borneol
Yarrowia Lipolytica is a safe oil-producing yeast that is considered to be an excellent host for the production of terpenoids because of its hydrophobic environment in the cell. The oil droplets in Yarrowia Lipolytica cells can effectively intercept the produced terpenoids to avoid toxicity to engineering bacteria. The high-throughput acetyl-CoA metabolism in cells provides sufficient raw materials for the production of terpenoids.
Peroxisome-cell compartment engineering strategy
Yarrowia Lipolytica has an endogenous MVA pathway in the cytoplasm, but this pathway is usually competed by the synthesis of other terpenoids such as squalene. Therefore, we attempted to reconstruct the MVA pathway in the peroxisome of Yarrowia to isolate it from similar metabolic pathways in the cytoplasm. This attempt has been proved effective.
At the same time, peroxisome is the place where yeast undergoes β-oxidation, and a large amount of acetyl-CoA is directly provided to the MVA pathway we constructed ; the narrow space of peroxisome limits the diffusion of enzyme and substrate, which further improves the production efficiency of borneol.
BbTPS3- Selection of efficient borneol synthase
Borneol is a monoterpene compound, which is directly processed by borneol diphosphate synthase from the common precursor GPP of terpenoids. At present, BPPS from various plant sources such as borneol camphor and sage have been found. We finally chose BbTPS3 from Blumea balsamifera as a synthetic enzyme for the production of medical borneol : this enzyme has extremely high chiral selectivity, and the produced ( - ) -borneol can account for 95.30 % of the total product, which saves the cost of purification for the next drug crosslinking and increases the safety of borneol as a drug. The heterologous expression of this enzyme has been verified in Saccharomyces cerevisiae.
MVA pathway- Rapid Construction of Complex Metabolic Engineering
In order to rapidly construct the mevalonate pathway in the peroxisome of Y.lipolytica, we constructed the following three plasmids :, which were carried with the MVA pathway-related enzymes and BbTPS3 initiated by the strong promoters pTEFin and pGAP in Y.lipolytica, and the C-terminus of each enzyme was added with a PTS1 sequence to target it to the peroxisome. Each plasmid carries the URA3 selection tag of Yarrowia lipolytica and the amp resistance tag of Escherichia coli for shuttle use between Escherichia coli and yeast. Before the yeast is transformed, the plasmid is linearized by restriction enzymes to facilitate insertion into the yeast genome break.
Module 2 :efficiently-produced Bor-Nps
Part Ⅰ Borneol modified nanoparticles function
Lipid nanoparticles (LNPs), are nanoparticles composed of lipids. They are a novel pharmaceutical drug delivery system (and part of nanoparticle drug delivery), and a novel pharmaceutical formulation.
BBB, a diffusion barrier essential for the normal function of the brain, impedes the entrance of substances from the blood to the brain to maintain brain homeostasis. But this has been the source of some challenges of brain drug delivery systems,including our LNPs.
At the same time borneol has been used widely in traditional Chinese medicine as a messenger drug, which facilitates the transport of multiple drugs to specific sites and harmonizes the effects of those drugs. Borneol can also significantly loosen the intercellular tight junctions (TJs) and increase the number and volume of pinocytosis in in vitro Blood-Brain Barrier(BBB) models. Borneol can affect BBB in three ways:
1. Borneol can reduce the TJ and degradation of the vascular basement membrane,increasing the BBB permeability by affecting Ca2+-eNOS-NO and VEGF-eNOS-NO signal pathways.
2. Borneol can make brain microvascular endothelial cells shrink, augmenting the gap between cells.
3. Borneol has the potential to adjust the function of permeability glycoprotein(Pgp) and increase the bioavailability of other drugs in the brain.
As mentioned above,Pgp is a specific type of ABC transporter that is found in epithelial cells of the BBB, extrudes hydrophobic substances out of the cell and depends on ATP for energy. Borneol can bind with the Pgp and decrease the transform function of Pgp, which helps reduce the excretion of our drugs. At the same time ,the defuctionalization of Pgp also contribute the accumulate of the 5--hydroxytryptamine (5-ht), an open transmitter of blood-brain barrier.
So if we load borneol on our nanoparticles, the borneol will help both open the BBB and inhabit the excretion of our drugs. Once our Bor-Nps enter the cell, these borneol will influence the Pgp ,the Ca2+-eNOS-NO and VEGF-eNOS-NO signal pathways through many cytokines and finally achieve a better drug delivery effectiveness.
Part Ⅱ Preparation of the Bor-Nps
The base unit of LNPs is methyl-poly(ethyleneglycol)–poly(lactic-co-glycolic acid) (mPEG-PLGA),often works as the framework material of the LNPs. But we can not load our borneol on mPEG-PLGA directly, for there is no site to form a chemical bond.
So we choose succinimide group of Nhydroxy succinimide–poly(ethyleneglycol)–poly(lactic-co-glycolic acid) (NHS–PEG–PLGA ) as the carrier. However, the active site of borneol is the hydroxide radical, but there is a mass of hydroxide radical on the PEG,and many side effects may occurred if we use borneol directly. So we turn borneol into the bornylamine to guarantee the uniqueness of reaction. And we designed the following Chemical process:
1. With acetone; zirconic acid In benzene at 80℃; for 8h;
2. With (pyridine)(tetrahydroborato)zinc In tetrahydrofuran for 1.5h; Heating;
3. With pyridine; hydroxylamine hydrochloride at 75℃; for 2h;
And we wish further grope and optimize the reaction condition in future.
The Bor-Nps was synthesized by incubating of NHS–PEG–PLGA with bornylamine at a molar ratio of 9:1 at room temperature for 24 h. After purification by methanol precipitation,we intend to identify the outcome with NMR.
