Measurement
Measurement
Through the introduction of multiple metabolic pathways in yeast, we succeeded in producing three MAAs molecules (shinorine, porphyra-334 and palythine) as well as gadusol, an alternative MAAs analogue. The absorption spectrum of these four molecules can cover almost the entirety of the UVA (315-400 nm) and UVB (280-315 nm) wavelength. This trait further indicated that our four molecules, used in a novel sunscreen, can provide a full range of protection against UV radiation.
Initially, we were unable to quantify the fermentation products of yeast due to the difficulty of obtaining standards of the four molecules from the market. However, in the process of optimizing the yeast for modification, we needed a standard to measure the yield. Therefore, we established a measurement method to quantify our samples in order to provide a reference and help future iGEM projects with solving the difficulty of gaining standards for testing.
Absorbing Scanning
Firstly, the four MAA molecules have different absorption spectrums and have maximum absorption peaks at various locations throughout the wavelength. Based on this property, we scanned our yeast fermentation products in the 280-400 wavelength band. Through determining the wavelength value at the maximum absorption peak, we identified the molecules' species; while examining the height at the maximum absorption peak also helped us to initially quantify the product relative to each other. Using this method, we confirmed the optimized protocol for the production and yeast modification for our four molecules.
In our experiments, we discovered that both shinorine and porphyra-334 have the maximum absorption value at 334 nm. For the production of shinorine and porphyra-334, we used different AGL and Alal for recombination to obtain the optimal enzyme combination. By scanning the fermentation supernatant of the yeast strain, we observed a clear absorption peak at 334 nm, which represents the successful production of shinorine or porphyra-334. By comparing the absorption peaks at 334 nm, we confirmed that Np5598 is the most efficient AGL and NlmysD is the most efficient ALAL (shown in Figure 1).
Figure 1: Absorption spectrum of the AGL-ALAL combinations of L5 strains that produces shinorine or porphyra-334. We scanned the OD of broth supernatant after 72 hours of fermentation of 8 groups of yeast (except for Am4257-Am4256 which failed to grow), which shows a clear absorption peak at 334nm (A). We compared of OD334 values of the samples, discovering that Np5598 seems to be the most efficient AGL, while NlmysD being the most efficient ALAL. We conducted the same experiment with the yeast lysate of the 6 most efficient combinations and found a even more significant absorption peak (C). The OD334 value of the lysate supernatant affirms our previous conclusion that Np5598 is the best AGL and NlmysD the best ALAL (D).
Palythine has a maximum absorption peak at 320 nm. By scanning the fermentation supernatant of this yeast, we learned that a distinct absorption peak was observed in the optimal palythine producing strain L6:Np5598-Np5597-NlmysH (shown in Figure 2).This shows that we have achieved palythine production.
Figure 2: Palythine production. After 72 hours of culturing, the absorption peaked at 320nm.
Gadusol has a maximum absorption peak at 290 nm.After obtaining the absorption spectrum graph of the supernatant broth after 72 hours of SC.L4 fermentation, a slight absorption peak was observed at around 290 nm. To better observe the absorption spectrum to better determine the existance of the gadusol, we subtracted the negative control curve from L4's absorption curve (Figure 3). This shows that we have achieved gadusol production.
Figure 3: Gadusol production. Fermenting the L4 strain, using the L2 strain as a comparison. After 72 hours of culturing, the absorption curve became clear, and it can be seen that L4 has a peak at 290nm (A). We compared L4’s values to the control, obtaining a clear absorption peak for gadusol.
Confirming shinorine and porphyra-334 production through HPLC
Although we have found the most efficient AGL-AlaL combinations through absorption scanning, we could not determine whether the output was shinorine, porphyra-334, or both. We learned through background research and HP activities that nori samples mainly contains the MAAs shinorine and porphyra-334, the target molecules in our study. Therefore, we used nori samples as control to determined our product of the yeast combinations using HPLC and MS.
We found that the metabolite of Np5598-NlmysD mainly has the absorption spectrum of porphyra-334 according to HPLC, and the result of MS also proved that mainly porphyra-334 exists in the metabolite (m/q = 347). This shows that NlmysD has a strong selective preference toward the amino acid Threonine, and thus will mainly produce porphyra-334 if both Threonine and Serine are present in the environment. Np5598-Np5597, on the other hand, shows shinorine's absorption peak, indicating its preference toward serine. Therefore, we decided to use Np5598-NlmysD for porphyra-334 production and Np5598-Np5597 for shinorine production (shown in Figure 4).
Figure 4: 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.
Extracting standard product from nori
Through our essay research and HP activities, we learned that nori, as a naturally occurring organism that contains MAAs, can be used as a raw material for the preparation of MAAs standards. Nori contains two main MAA molecules: shinorine and porphyra-334, which are also our main products. Therefore, we used nori extract as a standard for further quantification of these two products in yeast fermentation broth.
We used two methods for the extraction of nori (Zhang H et.al.,2022, Zhang mm,2015 ). By MS test, we discovered that the extract of method 2 (Zhang mm,2015) had a higher purity (shown in figure), so we chose this sample for the subsequent standard production (shown in Figure 5).
Figure 5: Two methods of nori extraction. Results varied methods based upon the aforementioned essay. MS test indicated the extract from method 2 had higher purity in extraction
We extracted 69 mg of MAAs lyophilized powder from 2.5 g of nori. We took 20 mg of the lyophilized powder and dissolved it in 1mL of water to obtain a MAA standard solution with a concentration of 20mg/ml, and we used this standard sample to establish the standard curve for measurement. We mainly quantified the fermentation broth of two strains, the porphyra-334 producing Np5598-NlmysD strain and the shinorine producing Np5598-Np5597 strain (shown in Figure 6). 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 6: standard curve for nori extraction based on the porphyra-334 producing Np5598-NlmysD strain and the shinorine producing Np5598-Np5597 strain. The shinorine titer of L7-Np5598-Np5597 is 138.8mg/L, and the titer of L8-Np5598-Np5597 is 258.6mg/L. The porphyra-334 titer of L7-Np5598-NlmysD is 210mg/L, and the titer of L8-Np5598-NlmysD is 270.7mg/L.