Team:UM Macau/Engineering
Engineering
Overview
Our project is to design a beer that is antioxidant and with low purine content, which is healthier and has a lower risk to get gout than the beer on the market. We constructed yeast with the genes of our interest, SOD1, PNP1, and chit42. To determine if we have constructed the modified yeast successfully, we performed function assays to confirm it.
Design
We aim to genetically manipulate the brewing yeast to endow it with the capacity to scavenge radicals, enhance the antioxidant ability of beer, as well as decrease purine content. Three distinct functions are achieved by introducing three genes individually to yeast to create a community of yeast to optimize beer quality. These three genes include SOD1, chit42, and PNP1.
Chitinase (chit42) encoded by chit42 breaks down chitin, one of the most predominant components of the yeast cell wall. chit42 has well been documented to catalytically decompose chitin into its oligomers: Chitooligosaccrides (COS), which can further quench the radical species and enhance the antioxidant ability of beer.
PNP1 encodes purine nucleoside phosphorylase. It is an enzyme that could decompose nucleotides into purine bases, allowing yeast to use purines more efficiently, thereby decreasing the concentration of purine in the fermentation broth.
Superoxide dismutase 1 (SOD1) is a significant antioxidant, and it is regarded as a fundamental defense agent against reactive oxygen species. It is a class of enzymes that removes ROS by converting the harmful ROS by catalyzing the disproportionation of superoxide ionic radicals (O2-) into H2O and O2. Through this principle, SOD1 can be a good scavenger of superoxide radicals in beer. In previous reports, SOD1 has been shown to reduce the amount of free oxygen in beer, which in turn increases the antioxidant activity of beer to maintain flavor stability.
Build
Our strategy for helping yeast acquire such versatile function is mainly based on the construction of three vectors carrying these three genes, and the yeast community is created by transforming genes into S. cerevisiae respectively. The cloning results for SOD1 and PNP1 are similar, both are cloned in pFA6a vector together with a 3 x HA tag, alpha factor, 6 glycines to balk self-coiling, and pGK1 promoter, all of which are flanked by ADE2 homologous sites. As to gene chit42, it is cloned into the same vector but rather linked with a TPS1 promoter. The pFA6a vector for cloning also contains the kanMX sequence which is for the screening test.
The first step of cloning is to proliferate the three fragments: ADE2-promoter-alpha-factor-gene-ADE2 in E. coli. The insert fragments are firstly extracted from the pUC57 vector by PCR, then the Gibson assembly is adopted to ligate the pFA6a plasmid linearized by primers targeting the ADE2 site with inserts. The E. coli is treated by heat shock and miniprep is performed to extract amplified pFA6a vector containing our inserts and gel purification is done to further purify the DNA construct.
The second step is to introduce the DNA construct into Saccharomyces cerevisiae FY4, after using PCR to extract a fragment from three constructed plasmids. This process is achieved by homologous recombination using the ADE2 site flanking the genes of interest. After that, further screening for positive cloned yeast is performed by streaking yeast strain onto YPD medium containing 400ng/ml G418, the counterpart antibiotic of kanMX sequence.
Figure 1. The wild-type strain (left) and SOD1 strain (right)
Figure 2. The wild-type strain (left) and PNP1 strain (right)
Figure 3. The wild-type strain (left) and chit42 strain (right)
The successfully introduced yeast grew a red colony on the plate, and the genetically modified yeast can be used to examine the protein's activity by doing the function assay:
After culturing the genomic modified strain and FY4 (the wild-type strain) strain using YPD media overnight, we filtered the centrifuged supernatant of the medium with a 0.22um filter to obtain a yeast-free medium. These mediums were used to test the activity of SOD1, PNP1, chit42 later. Specifically, we used pyrogallol assay to test the function of SOD1 strain, DPPH assay to test chit42 strain, and Purine Nucleoside Phosphorylase Activity Assay to test the function of PNP1 strain.
Test 1. Pyrogallol Assay
Pyrogallol is the chemical reagent that can autoxidize in the presence of molecular oxygen as it serves both as the source and as a scavenger of oxygen-free radicals. It is often used to detect the enzyme activity of SOD1 by the inhibition rate of SOD1 to its self-oxidation. In our project, we also used pyrogallol to evaluate the activity of SOD1 that our genetically modified strain produced.
Under the condition of the alkaline solution containing EDTA, we mixed pyrogallol and water, with YPD Media grown by SOD1 strain, and YPD medium grown by the wild-type strain, respectively as the test group. At the same time, we also made a mixture without pyrogallol as blank. After mixing, they were quickly divided into 96-well plates. Each tube of sample is added to three Wells. During the following half-hour, we measured the absorbance value of the solution at the wavelength was 325nm every minute until 30 minutes.
According to the change of absorbance in unit time of the control group and experimental group, we got a pyrogallol autoxidation rate diagram. By comparing the slope difference between the wild-type strain and SOD1 strain curves, we can know the difference in their inhibition rate of pyrogallol autoxidation
It is worth mentioning that before we did the Pyrogallol function assay of the medium of strain, we first used a known antioxidant vitamin C as a positive control to test the effectiveness of pyrogallol.
Figure 1
According to this result, ascorbic acid concentration above 0.1mg/ mL can effectively inhibit pyrogallol autoxidation. Therefore, our Pyrogallol function assay can be proved to be meaningful.
Figure 2. Pyrogallol function assay of SOD1 strain and the wild-type strain. And we add H2O in the self oxidation group as control.
According to this result, we found that the medium of SOD1 and the wild-type strain both had a strong effect on pyrogallol self oxidation, but there was not much difference between SOD1 and the wild-type strain. In order to further verify this result, we decide to change H2O into YPD medium in the self oxidation group to determine whether YPD medium itself has an impact on Pyrogallol auto-oxidation.
Figure 3
Figure 3 shows the result of pyrogallol assay at wavelength 320 nm after changing H2O into YPD medium in the self oxidation group. We found that the slope difference between the wild-type strain group, the SOD1 group, and the self oxidation group was very small, which in fact could not effectively prove that the SOD1 group had a better inhibitory effect on Pyrogallol autoxidation.
In order to improve the pyrogallol assay, we used the 30kDa centrifugal filter devices to filter our medium (the size of SOD1 is 29.8kda) in the following experiment. The benefits of doing this are mainly in two aspects: 1. The concentration medium increases the concentration of our target protein. 2. Filter out some small miscellaneous proteins to reduce the influence of background.
Pyrogallol assay after we use the 30kDa centrifugal filter devices to filter our medium.
Figure 4
Figure 5
Figures 4 and 5are the results of the Pyrogallol assay after the YPD medium was filtered by the 30 kDa centrifuge. In particular, we also tested pyrogallol at 420 nm wavelength, because the oxidation product xx of Pyrogallol also has a strong absorption peak at 420 nm wavelength. At this wavelength, the absorption peak of the YPD medium is smaller, which can better exclude the influence of the YPD medium on the experiment itself. However, the slopes of the SOD1 strain and the wild-type strain were still almost the same.
Considering the high absorption peak of Yeast Extract-Peptone-Dextrose (YPD) medium in solution and the inclusion of yeast extract in YPD media. We decided to replace the YPD medium with the synthetic complete (SC) medium to culture our strain. The reason we choose to use the SC medium is that, firstly, the absorption peaks of the SC medium were much smaller in the 325 nm wavelength used in our pyrogallol assay. Secondly, the components of SC Media are all known. Compared with YPD containing yeast extract, it may have less background influence on the experiment itself. The below figures show the composition difference between SC medium and YPD medium.
Before we officially started the second round of function assay, we did two things to ensure the necessity and validity of replacing with the SC medium.
Firstly, the absorption peaks of YPD and SC medium at 325 nm and 420 nm were compared and measured. According to this result, we can know that the problem of background absorption peaks is basically solved well.
Secondly, we detected and compared the difference of growth rate between the wild-type strain and Genetic Modified Strain in the original YPD media and the newly replaced SC Media.
Figure 6
Figure 7
By comparison, we found that the YPD medium and SC medium had little influence on the growth speed of the wild-type strain. However, for GM Strain, their growth rate in SC media is much less than that in the YPD medium.
This pre-experiment provided convenience for our subsequent Function assay because according to this growth curve, we roughly inferred in the following Function assay how long we should culture our Strain in advance to make it reach a better state.
We did the pyrogallol assay again, and this time we used SC medium to culture yeast.
Figure 8. Pyrogallol assay after changing the medium of culturing yeast from YPD medium to SC medium that added a group of the SC media without yeast was added as control.
According to the result above, we found that the concentrated medium still had a certain light absorption value under our experimental wavelength. And considering that the 30 kDa filter we used before may not be able to concentrate our protein effectively because the size of our protein is 29.8 kDa and the 3D structure formed by it, we speculated that it might leak out from the gap of the filter. So we used the new 10kDa centrifugal filters to concentrate the strain medium.
Pyrogallol assay after using the 10kDa centrifugal filters to concentrate the strain medium.
Figure 9
Figure 10
Figure 9 and 10: Pyrogallol assay after changing the medium of culturing yeast from YPD medium to SC medium and using a 10kDa filter to concentrate the medium. overall result: Combined with the results of our six or seven function assays, we did not successfully prove the efficacy of SOD1. Although there was a certain difference between the SOD1 strain and the wild-type strain in the results of the function assay, we found that the difference was not stable after repeated experiments, and it was considered as an error of the experiment itself. As for the reason for this result, we speculate that it may be related to the insufficient secretion of SOD1. Due to the limitation of time and experimental environment, we have no way to expand the cultivation of yeast and the scale and time. For future experiments, we plan to enhance the simulation of yeast fermentation conditions to some extent and hope that it will increase the antioxidant capacity of the SOD1 strain.
Test 2. Purine Nucleoside Phosphorylase 1 Activity Assay
PNP1 is an enzyme that could break down the inosine to hypoxanthine, the developer could convert the hypoxanthine into urea acid. Urea acid is measured at a wavelength of OD =293nm. We add the supernatant of fermentation broth into the reaction mix, then measure the OD at 293nm every two minutes for 30 minutes, the slope of the graph will show the activity.
We add the inosine and developer into the PNP1 assay buffer at room temperature to form a reaction mixture. Add the supernatant of the wild-type strain and PNP1 strain respectively as the test groups, at the same time, prepare exactly the same reagents except for the developer as the background group. Dividing into U.V. transparent plate(96-well). Then measure the absorbance at OD=293nm every two minutes for 30 minutes. Compare the slope of the test group, we can know the activity of PNP1 or whether PNP1 is effective or not.
In the assay process, we found the slope is negative instead of positive as we predicted. (Figure 1)
Figure 1. PNP1 activity assay curve
Figure 2. Standard test curve
All the test groups and background groups have a negative slope, and the gap between them is too small. PNP1 might have no activity during the test, and we will state our hypothesis and possible solution in the following learn and redesign part.
Test 3. DPPH Assay
The DPPH radical scavenging assay is performed by using DPPH assay, (Chen et al. 2014). Prior to the real test, the chit42 yeast and the wild-type strain are incubated in SC medium overnight until the OD reaches 0.5-0.7. Then, 8% ethanol was added to induce the production, and the yeast was incubated under the same circumstances for another 18 hours: 25 degrees and rotating speed at 220 rpm. Before the examination, yeast was centrifuged, the supernatant was filtered to do the DPPH test. Briefly, 50ul samples with the addition of 200 ul 51.54 mg/L DPPH solution in 95% ethanol are labeled as As. Besides, Ao is by mixing 50 ul ethanol with 200ul DPPH solution of the same concentration. Additionally, Ar is made by adding 50 ul samples to 200 ul 95% ethanol. During the test, the three groups are triple-examined by using 96 microwell plates. Each group is incubated at 25 degrees in darkness for 30 minutes. After that, all three groups in the same well plate examined the light absorbance are performed by light spectrophotometry at 517 nm. And the DPPH radical scavenging rate of each sample can be deduced by the equation below:
DPPH radical scavenging rate= (1-(As-Ar)/A0)*100%
Figure 1. DPPH function assay of chit42 with ethanol concentration: 8%
The FY4 strain modified by chit42 containing vector shows enhanced ability to remove free radicals in solution, as a comparison with the wild-type strain. The result demonstrates that the TPS1 pomoter_chit42_alpha factor part works.
In combination with the western blot result that chit42 also shows positive results when ethanol concentrations are 2%, 4%, we diminish the ethanol amount to induce endochitinase production. After the wild-type strain and chit42 are both induced by 4% ethanol, the result illustrated in figure 2 indicates a higher efficiency regarding the DPPH scavenging rate of 4% induced chit42 strain as a comparison with the same strain induced by 8% ethanol. Considering the referential ethanol concentration from literature, we consider the amount of ethanol we add to reach 8% v/v ethanol actually interfere with yeast growth or activity. We hypothesize that yeast may self-produce some ethanol which is sufficient to induce enzyme secretion because of the oxygen exhaustion during culturing, and due to the time limit, we haven’t confirmed the best ethanol inducible concentration of ethanol. The actual optimal concentration may lays between 0 and 8%. Despite this, the result of 4% induction and 8% induction still illuminate the efficacy of radicals scavenging activity of chit42 modified yeast.
Figure 2. DPPH assay for 4% ethanol induction of chit42 yeast.
Reference
Yang, F., Luan, B., Sun, Z. et al. Application of chitooligosaccharides as antioxidants in beer to improve the flavour stability by protecting against beer staling during storage. Biotechnol Lett 39, 305–310 (2017). https://doi.org/10.1007/s10529-016-2248-3
Kidibule PE, Santos-Moriano P, . Use of chitin and chitosan to produce new chitooligosaccharides by chitinase chit42: enzymatic activity and structural basis of protein specificity. Microb Cell Fact. 2018 Mar 22;17(1):47. doi: 10.1186/s12934-018-0895-x. PMID: 29566690; PMCID: PMC5863366.
Chen C, Tong Z, Liao D, Li Y, Yang G, Li M (2014) Chemical composition and antimicrobial and DPPH scavenging activity of essential oil of Toona sinensis (A. Juss.) Roem from China. Bioresources 9:5262–5278