Drinking alcohol is enjoyed by people everywhere, however, excessive alcohol consumption can lead to alcohol poisoning and the harmful effects of other substances. It is alarming that such widespread activity can potentially threaten human health. To address this issue, we have employed technological means to eliminate this threat and make drinking safer for individuals.
In our experiments, we introduced the AocI gene into yeast, enabling it to break down biogenic amines produced during fermentation. We have also designed a process utilizing the FAD gene to enhance the expression of this enzyme. As a result, our product - the amine-reducing brewing yeast - produces alcohol-free from the threat of biogenic amines.
Engineering Construction Flowchart
Design
pAO815 is a frequently used isotopic carrier in Pichia pastoris. It serves as a vehicle for expressing the target protein within the yeast. AocI is the main part of our design which will express amine oxidase so as to reduce the amine level during yeast fermentation. Thus our first design to construct the plasmid pAO815-AocI.
We will prepare PCR templates for the target genes AocI and the linear pAO815 vector, then perform separate PCR amplifications for these two fragments. The amplified fragment was then used to synthesize the pAO815-AocI plasmid. Then, the groups of plasmids were transformed into DH5α cells for more copies.
Build
We ordered gene templates of AocI from the company Genscript and the vector is provided by SubCat. Homologous recombination was performed to construct the plasmid pAO815-AocI with enough gene fragments and following that Heat shock was processed to transform the plasmid into the competent cells for further cultivation.After second recycle, the plasmids were further added into our host yeast GS115.
Figure 1A depicts the agarose gel electrophoresis results of PCR amplification, showing that the observed bands match the expected size (2285 bp) of the PCR product, AocI. In Figure 1C, the alignment results of the gene fragments indicate a perfect match with the desired sequences of gene AocI, showing 100% identity.
Figure 1. The successful construction of recombinant plasmid pAO815-AocI.
Test
After product purification, we used protein electrophoresis to verify whether the major protein AocI was successfully induced in culture and checked whether AocI was successfully expressed.
As shown in Fig 2, protein AocI has a size of 85kDa. There was a clear difference between the protein band of yeast containing the AocI plasmid and the blank control group, indicating that the AocI gene was successfully expressed.
Figure 2. Testing the existence of AocI protein in pAO815-AocI.
Bromocresol purple, as an acid-base indicator, turns yellow in acidic conditions and purple-red in alkaline conditions. Therefore, after adding histamine to the system, the culture medium will exhibit a purple-red color. When histamine is hydrolyzed by oxidases, the products are ammonium salts and aldehydes, which are weakly acidic. As a result, the alkalinity in the hydrolysis area decreases, gradually changing to yellow. Thus, it is possible to determine whether histamine has been oxidatively hydrolyzed by observing the color change.
In order to evaluate the activity of amine oxidase expressed by our yeast, bromocresol purple and different histamine concentrations were added to the PDA solid medium.The specific instructions are as follows: bromocresol purple, ampicillin (that is, antibiotics), and histamine with different concentrations were added to these five Petri dishes, and the concentrations were 0 μ g/ml, 20 μ g/ml, 50 μ g/ml, 100μ g/ml and 200μ g/ml respectively. Then, the PDA was placed in four areas of five Petri dishes, and the Petri dishes were sealed. Two regions were GS115 blank, and the other was pAO815-AocI so that they could stand for one night.
As shown in Figure 3, a yellow bigger circle appeared in pAO815-AocI plate compared to the wild which indicates that we have successfully cracked biogenic amines into amines and aldehydes under the catalysis of amine oxidase.
Figure
3.
The results of the pAO815-AocI transparent circle experiment and the control group.
In order to further investigate the ability of the strain to degrade histamine, we also performed an HPLC assay. Histamine concentrations were measured at five different temperatures (20, 25, 30, 37, 45℃) and different sampling times (12, 24, 36, 48, 72 h).
We first incubated the bacterial solution to OD 0.6-0.8 to give the bacteria the highest viability. Then histamine was added and the initial concentration of histamine was 200ug/ml. changes in histamine content were detected using HPLC. As can be seen from the chromatogram in Fig 4, compared with the control group GS115, pAO815-AocI, the measured time of histamine peak was around 18-19 min. According to the calculations, it was learned that the concentration of histamine was significantly decreased after 72h of continuous cultivation.
Figure 4. The HPLC results of the pAO815-AocI HPLC experiment and the control group.
According to the image results, the content of histamine decreased gradually with time, and the fastest degradation of histamine was observed at 30℃. The lowest content of histamine was observed at 72h.
Figure 5. Results of histamine concentration over time.
Learn
In this part of the experiment, we learned how to construct a plasmid and use a PCR device. Even though most of the process went smoothly, we still encountered some problems. For instance, one of the PCR results didn’t turn up well, and the performance of some metrics was poor in the subsequent tests, but fortunately, most of the product is available for later use.
In addition, we also studied how the methanol concentration of induction affect the protein expression, and we can basically draw the conclusion that the highest expression level is shown in Figure 6, indicating that 0.50% is the optimal induction concentration. For further analysis, mathematical model will be applied to dig into these data.
Figure 6. The results of testing the existence of target protein under multiple concentrations of methanol.
Design
In order to promote the expression of AocI, we integrated the plasmid pAO815-AocI with another gene FAD which could express histamine dehydrogenase to facilitate amine oxidase. The same protocol will be proceeded based on the FAD template and pAO815-AocI.
Build
We ordered gene templates of AocI from the company Genscript. Homologous recombination was performed to construct the plasmid pAO815-AocI-FAD with enough gene fragments and following that Heat shock was processed to transform the plasmid into the competent cells for further cultivation. After second recycle, the plasmids were further added into our host yeast GS115.
Figure 5A shows the agarose gel electrophoresis results of the gene fragment, as indicated in the legend. In Figure 5C, the alignment results demonstrate a high similarity between the observed gene fragment and the expected sequence. Precisely the inserted gene fragment matches the desired sequence. *The area we requested to sequence covered the target region, so in fact the target region alignment is 100%.
Figure 7. Construction of recombinant plasmid pAO815-AocI-FAD.
Figure 8C shows the yeast colonies that have grown after cultivation. Figures 8A and 8B display the experimental results of the pAO815-AocI-FAD insertion into the vector pAO815 linear, indicating a highly successful construction step.
Figure
8.
A: PCR testing result of plasmid pAO815-AocI-FAD linear
B: PCR testing result of plasmid pAO815-AocI-FAD linear in GS115
C: GS115 colonies containing plasmid pAO815-AocI-FAD in the SDS-His medium
Test
Figure 7 illustrates the experimental results of SDS-PAGE gel electrophoresis under different methanol concentrations. The experimental samples were treated with various methanol concentrations to induce yeast protein expression. The red portion in the gel represents the target protein, and successful practical outcomes are indicated by the blue staining with Coomassie Brilliant Blue, as shown in the blue-colored regions. Protein AocI has a size of 85 kDa and compared with the marker, we can see that there was a clear difference between the protein band of yeast containing the AocI plasmid and the blank control group, indicating that the AocI gene was successfully expressed.
Figure 9. Testing the existence of AocI protein in pAO815-AocI-FAD.
In order to evaluate the activity of amine oxidase, transparent circle assay with bromocresol purple was conducted with the wild GS115 as the control. As shown in figure 8, a yellow bigger circle appeared in pAO815-AocI-FAD plate compared to the wild which indicates successful engineering for amine oxidase coded by AocI.
Figure 10. The results of the pAO815-AocI-FAD transparent circle experiment and the control group.
To further evaluate the engineering of FAD part in pAO815-AocI-FAD, we compared these two groups and it presented tha the group pAO815-AocI-FAD (Figure 11-B) has bigger yellow circles than the group pAO815-AocI (Figure 11-A) at the same given concentration of amine.
Figure 11. A: group pAO815-AocI transparent circle experiment with the control; B: group pAO815-AocI-FAD transparent circle experiment with the control group
In order to further investigate the ability of the strain to degrade histamine, we also performed an HPLC assay. Histamine concentrations were measured at five different temperatures (20, 25, 30, 37, 45℃) and different sampling times (12, 24, 36, 48, 72 h).
We first incubated the bacterial solution to OD 0.6-0.8 to give the bacteria the highest viability. Then histamine was added and the initial concentration of histamine was 200ug/ml. changes in histamine content were detected using HPLC. As can be seen from the chromatogram in Fig 9, compared with the control group GS115 and pAO815-AocI-FAD, the measured time of histamine peak was around 18-19 min. According to the calculations, it was learned that the concentration of histamine was significantly decreased after 72h of continuous cultivation, and the pAO815-AocI-FAD strain had a more significant decrease in histamine concentration than the pAO815-AocI strain, which indicated that the pAO815-AocI-FAD strain had a better ability to degrade histamine, and this is in line with the result of the previous clear circle experiments.
Figure 12. The results of the pAO815-AocI-FAD HPLC experiment and the control group.
According to the image results, the content of histamine decreased gradually with time, and the fastest degradation of histamine was observed at 30℃. The lowest content of histamine was observed at 72h. A pattern can be observed: all show monotonically decreasing, but obviously, the rate of decrease at 30℃ is accelerated, and also after 73h of experiment, the lowest values.
Figure 13. Results of histamine concentration over time.
Learn
From the experiment, we learned how to transform yeast. At the same time, we also experienced some difficulties. For example, the concentration of PAF plasmid was not enough due to the first addition, so the amount of plasmid added for the second time increased, which made it easier to see the results of subsequent protein expression. Meantime we also studied how the methanol concentration of induction affect the protein expression, and we can basically draw the conclusion that the highest expression level is shown in Figure 6, indicating that 0.50% is the optimal induction concentration. For further analysis, mathematical model will be applied to dig into these data.
Figure 14. The results of testing the existence of target protein under multiple concentrations of methanol.
After the engineering success in Pichia pastoris host, it is necessary to transform the vector into Saccharomyces cerevisiae for implementation tests since Saccharomyces cerevisiae is more commercially used in alcohol production industry. Therefore, there are more engineering cycles to do with the new host, Saccharomyces cerevisiae. After team discussion with the instructor, we have drafted the preliminary design of the plasmid construction with another vector, pYES for Saccharomyces cerevisiae use.
Figure 15. Construction of recombinant plasmid pYES-AocI.
Figure 16. Construction of recombinant plasmid pYES-AocI-FAD.
In the future, we also hope to promote this technology to wineries and food factories so that people can enjoy healthier drink and food.