Team:USTC

Achievement

plasmid construction

    From the wild-type Y.lipolytica strain W29, we obtained the coding sequence of the enzyme in MVA pathway by PCR and attached PTS1(GGGSSKL) to the C terminal when primer was designed so that our enzyme can be located in the peroxisome.Then, we used Vazyme ClonExpress II One Step Cloning Kit to complete the construction of single-gene plasmids. Afterwards Colony PCR confirmed the successful insertion of the coding sequence, and the results of Sanger sequencing were the same as expected.(Figure1)

Figure 1. 1% agarose gel electrophoresis of the core enzyme gene of MVA pathway.

    At the same time, in order to construct the MVA pathway in Y.lipolytica more quickly, we constructed double-gene plasmids by restriction enzyme digestion. Then, the results of Sanger sequencing in coding region confirmed that we successfully constructed double-gene plasmids (BBa_K4231017).

    In addition, in view of the limited available tag in Y.lipolytica, we try to improve the utilization rate of tags,which means using a tag to carry more enzyme genes into Y.lipolytica. Therefore, we tried to construct triple-gene plasmids (BBa_K4231019) and quadri-gene plasmid (BBa_K4231018). Thanks to Takara Infusion cloning, our attempts were successful and complete the PvuII restriction enzyme digestion identification and Sanger sequencing test.

Figure 2. The restriction enzyme digestion results of three main parts.

tag recycling

    We used a variety of means to make the most of the limited available tags in Y.lipolytica. We successfully constructed pUC19 plasmid backbone carrying HisG-URA3-HisG ( BBa_K4231020 ) and successfully finished enzyme digestion identification.

Figure 3. The restriction enzyme digestion results of pUC19-HUH.

    Unluckily, after we transformed the plasmid into the DH5a strain for amplification, we found that the plasmid was highly unstable and the recombination generated between the large homologous arms. But fortunately, in the process of our in-depth collaboration with NNU-China, we obtained the plasmid containing HUH from NNU-China. The identification results were promising.

Figure 4. The identification results of plasmid given by NNU-China.

Figure 5. The assemble results of pUC-SCP2-ERG10-ERG13-HUH-TE whose plasmid backbone is given by NNU-China, which is identified by restriction enzyme PvuII.

    Moreover, we successfully designed and constructed the knockout box for URA3 tag (BBa_K4231011). In practice, we found that high dosage knockout box and multiple knockout can achieve good results. After a large number of single colony screening on FOA selective medium, the knockout efficiency was higher than expected.

Figure 6. 2% agarose gel electrophoresis of knock-out box ( 400 bp ).

    Additionally, we also designed and tried to transform S.cerevisiae recombinase to improve the rate of homologous recombination. Specific explanation present in Engineering success.

regulatory element

    We have also successfully constructed oleic acid-induced mixed efficient regulatory sequence ( BBa_K4231016 ) by overlap PCR. This element is of great significance for our future work.

Figure 7. The overlap PCR results of Activator1-Regulator1 ( 600 bp ) and Activator1-Regulator1-Activator3 ( 938 bp )

bacterial strain

    After the construction of the MVA pathway in the peroxisome of Y.lipolytica was completed, we used the method colony PCR to ensure that each enzyme gene was successfully transformed and could stably exist in the chassis strain. Fortunately, we got the gratifying results. (Figure 8) All the enzyme genes were successfully transformed into Y.lipolytica.

Figure 8. All the eight enzyme genes of MVA pathway was identified by colony PCR. Among them, ERG19 and NADH-HMG were negative, which were successfully identified by normal PCR after exacting the genome of Y.lipolytica PYB1.

output

    After the chassis strain was completely constructed, we expanded it in a conical flask and fermented it for 72-96 hours. The fermented yeast was extracted with ethyl acetate and the target product borneol was identified by Gas Chromatographic Mass Spectrometer (GCMS). We got delightful results.

Figure 9.The identification result of borneol.

future plan

    At present, there is still much room for improvement in the production of borneol by our chassis strains, which limits our progress towards industrial fermentation. In future work, we plan to continue various engineering modifications of our chassis strains, such as : overexpression of fatty acid beta oxidation pathway, expression of terpenoid transporters and ATP transporters on peroxisome membranes, transformation and expression of a phosphatase, etc. We will try these modifications and further test the effect of such modifications on terpenoid production. Currently, due to the limited experimental time, Bor-NP have not carried any drugs. In the future, we plan to find a variety of drugs that exert efficacy in the brain, and try to embed them in Lipid nanoparticle to further test their efficiency of crossing the blood-brain barrier(BBB) to confirm that our project purpose can be achieved. Furthermore, in consideration of the superiority of Y.lipolytica to produce various terpenoids, we also plan to try to produce other monoterpenes, such as limonene, caryophyllene, zingiberene, etc., to expand the application prospect of our project.

borneol conversion

    We calculated the theoretical yield of borneol converted to borneol amine and connected to NHS-PEG-PLGA, which is about 73.93% (Figure 9) and meet our expectations.

Figure 9. yield of BO-NHS-PEG-PLAG

crossing the blood-brain barrier

    We modeled the diffusion of lipid nanoparticles in the human body and predicted the proportion of lipid nanoparticles passing through the blood-brain barrier based on the classical atrioventricular model (Figure 10). The model shows that while 1.5g of lipid nanoparticles are administered intravenously into the body its concentration in cerebrospinal fluid can reach 3.8385×10^(-3) g/L under the modification of borneol amine. This has played a role of reference and guidance for our future plans.

Figure 10. the relation between concentration of lipid nanoparticles crossing the blood-brain barrier and time