Proof of Concept

Abstract

In order to produce Mycosporine-like Amino Acids (MAAs), LINKS_China established multiple genetic circuit in order to produce shinorine, porphyra-334, palythine, and gadusol. By comparing High Performance Liquid Chromatography (HPLC) and Mass Spectrometer (MS) of nori samples containing both shinorine and porphyra-334 with our produce, we determined that we had successfully found the genetic circuit to produce shinorine and porphyra-334. After all 4 molecules are produced, we compared the absorption spectrum of our molecules, and comparison with results from papers show that we had successfully produced the correct MAA molecules. To determine the effects of MAA molecules in absorbing UV, we measured the strength of broth supernatant containing MAAs using UV test paper. We found there is a significant decrease in UV strength after MAA is used.

Figure 1: The production of MAAs. We introduced metabolic pathways of gadusol (λ(max)=290 nm), porphyra-334 (λ(max)=334 nm) ,shinorine (λ(max)=334 nm) and palythine (λ(max)=320 nm) in yeast (A).

Production of Shinorine and Porphyra-334

Previously, in our SC.L3 strains, we had succeeded in increasign S7P concentration and converted S7P to 4-DG. To produce shinorine and porphyra-334, we constructed 9 different combinations of ligases AGL and AlaL, which converts 4-DG into shinorine or porphyra-334. By performing High Performance Liquid Chromotalogy and Mass Spectrometer tests, we decided on two combinations, Np5598-Np5597 and Np5598-NlmysD which is responsible for the production of shinorine and porphyra-334, respectively.

Figure 2: High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) test of Np5598-NlmysD and Np5598-Np5597 compared with nori samples. Shinorine and Porphyra-334 lack a standard on the market, thus we decided to extract pure MAA from nori (Porphyra spp.) to use as standard. HPLC results (A) and MS results (B) show a peak in shinorine and porphyra-334, which proves that there is high concentration in nori extractives, making it a reliable standard. In Np5598-NlmysD’s fermentation broth, HPLC results (C) and MS results (D) show that mostly porphyra-334 exists. However, in Np5598-Np5597’s liquid, HPLC (E) and MS (F) results show mostly shinorine.

We used plasmid expression (L7 series) and genome inserting (L8 series) to express the two combinations of ligases. After fermentation for 72 hours in YPD medium, we compared the OD 334 absorption of the broth's supernatant between SC.L7 series and SC.L8 series. We found that the OD value of L8 series with genome recombinant, showed a steady increase of approximately 20% compared with that of L7 series with plasmid expression for both Np5598-NlmysD and Np5598-Np5597. Finally, the nori extract with a higher purity of shinorine and porphyra-334 is used as the standard sample to obtain rough yield data of SC.L8 series: the shinorine’s yield reached 258.5mg/L in L8-Np5598-Np5597, the porphyra-334’s yield reached 270.7mg/L in L8-Np5598-NlmysD.

Figure 3: Genome insertion of Np5598-NlmysD and Np5598-Np5597. To further increase and stabilize Shinorine and Porphyra-334 production, we inserted the genes Np5598-NlmysD and Np5598-Np5597 into the yeast’s genome at position 106 of chromosome I (Amanda R. et.al. 2017). After OD scanning, we found that the absorption peak of after genome insertion is higher than plasmid vector (A). We compared the OD334 value of plasmid transformation and genome insertion and found that there is about 20% increase in genome insertion expression.

Verifying Absorption Spectrum of MAA Produce

After we successfully produced shinorine, porphyra-334, we built onto the original circuit in order to produce palythine and gadusol. By comparing the absorption peak of our samples and those in papers, we affirmed the production of all 4 MAAs.

Figure 4：Comparison of MAA production. Through reading documents, we discovered the molecules gadusol, palythine, shinorine, and porphyra-334, which are able to absorb UV light within a range of 275 to 360 nm, absorbing UVB and the majority of UVA (A). After the fermentation of our yeast strains, it can be seen there are significant absorption peaks from 280nm to 360nm, perfectly conforming to our designs.

Testing the Ability of MAAs to Absorb UV

In order to test whether our MAAs have the ability to absorb UV as suggested in papers, we compared the strength of UV radiation on UV strength test paper. We selected porphyra-334, which has the highest yield among the MAAs, as samples. We used the broth supernatant of yeast porphyra-334-producing yeast strain containing Np5598-NlmysD after 72 hours of fermentation under YPD culture medium. After the samples dried, we shoned UV light mainly in the form of UVB onto the test paper for 10 seconds. It was seen that after porphyra-334 were added onto the test paper, the color after UV was shone onto the test paper was slightly lighter compared to when MAA was absent. This indicates that MAAs were able to absorb certain amounts of UV radiation.

Figure 5: UV absorption ability test of our yeast product. Drop of samples (A). Irradiated with UV light (mainly UVB) after samples were dried (B). Result of color development, the lighter the color, the less ultraviolet transmission (C).

Sunburn Repair

To achieve the aim of having skin-repairing, anti-aging and anti-inflammatory functions, we introduced SOD and catalase, fusing them together to eliminate ROS. We have assessed the enzyme activity of SOD and catalase via ELISA; kit detection showed the enzyme activity of sod reached 3.00U/mg, and catalase reached 128.84U/g

Figure 6: Design and detection of hSOD, hCatalase, and hSOD-flexible linker-hCatalase (SHC). We use promoter pT7 and vector pET28a to express hSOD, hCatalase and SHC and transformed these plasmids into E. coli (A). After inducing the expression of this gene, we used test kits to measure the enzyme activity of hSOD and hCatalase, respectively (B). The results of SOD enzyme acitivity test is shown (C) (Note: a lighter yellow indicates more enzyme activity). We then calculated the WST-8 formazan concentration after the reaction (D). Both figures shows that SHC is better at removing ROS than both hSOD and hCatalase. The results of Catalase enzyme activity test is shown (F) (Note: a lighter red color indicates more enzyme activity). The hydrogen peroxide content was then calculated (F) and it was shown that SHC is most efficient among the three enzymes.

Under the same conditions, we measured the enzyme activity of the fusion protein hSOD-linker-hCatalase (SHC), kit detection showed the SHC reached 8.05U/mg of SOD enzyme activity, and reached 313.04U/g of enzyme catalase activity. Therefore, the fusion protein has better performance than the two single proteins.Xanthine oxidase can catalyze the generation of ROS from xanthine. By adding fusion protein to the reaction, the generated ROS and H2O2 can be completely degraded after 60min of reaction.

Figure 7： Effects of hSOD, hCatalase, and SHC fusion protein in removing H2O2. In order to test the enzyme activity of hSOD, catalase and SHC in converting hydrogen peroxide into water and hydrogen, we tested H2O2concentrations. If catalase activity, peroxidase activity decreases, thus showing a less red color. The samples were tested after incubation for 30 minutes, and the color was recorded (B). A lighter color represents higher enzyme activity. The standard curve of H2O2 concentration to absorption was found (C). Using the standard curve, we calculated the remaining levels of H2O2 in the sample after catalase and SHC were added (D). SHC was shown to have greater enzyme activity.

We intended to use liposome in our product application, to wrap enzymes and play a role of deliver to transport enzymes into tissues and cells.We have obtained abundant suggestions on practical usage of our product by interviewing and inquiring medical professionals dedicated to ROS researchers: that we could engineer stem cells to produce this fusion protein, and thereby induce the stem cell to produce exosomes that are, in comparison to artificial liposomes, easier to be absorbed by our integumentary system. We aim to harvest and further mature our products by realizing this suggestion.

To know more details, please view our engineering page.