Team:SHSID/Proof Of Concept

SHSID

Proof of Concept

Overview

To solve the problem of insufficient supply of tropane alkaloids and reduce ecological damage, Our team fēvere factory developed genetically engineered yeast to produce putrescine, which can be chemically modified to produce tropane alkaloids. We genetically engineered two pathways of yeast to produce putrescine in order to enhance its efficiency and productivity. We improved the pathway that the yeast already has by increasing the gene expression of SPE1, which is responsible for the production of putrescine. We also inserted the AsADC and SPEB genes from oats into yeast to construct another more efficient pathway to produce putrescine. The plasmid containing three genes was inserted into the yeast, and its effectiveness was tested by LC-MS/MS.

Regarding future application or product, we consider producing putrescine efficiently and economically in the first stage, while in the second stage, we consider using our putrescine to synthesize tropane alkaloids.

Supporting Experiment Results

Plasmids containing target genes, AsADC, SPE1, and speB are successfully synthesized from 10 DNA segments. Segments 0 to 2 contain promoter PPGK1, target gene AsADC, and terminator TADH1. Segments 3 to 5 contain promoter PTEF1, target gene SPE1, and terminator TTEF1. Segments 6 to 8 contain promoter PTDH3, target gene speB, and terminator TCYC1. Segment 9 is the backbone plasmid pYES2. Transforming this plasmid into yeast will increase the yield of putrescine.

The first round of PCR
The first round of PCR amplifies DNA segments 0 to 7 separately.

Figure 1: Gel electrophoresis diagram. Lane 1 is segment 0. Lane 2 is segment 2. Lane 3 is segment 3. Lane 4 is segment 4. Lane 5 is segment 5. Lane 6 is segment 6. Lane 7 is segment 1. Lane 8 is segment 7.

On this gel electrophoresis diagram, all DNA segments were successfully amplified, and the band is on the corresponding position to its number of base pairs.

The second round of PCR
The second round of PCR overlapped two DNA segments from 0 to 7.

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Figure 2: Gel electrophoresis diagram. Lane 1 is segments 0 and 1. Lane 2 is segments 2 and 3. Lane 3 is segments 4 and 5. Lane 4 is segments 6 and 7.

This result shows that the overlap PCR between two DNA segments was successful because the bands are in their right position corresponding to the number of base pairs and relative to the ladder.

The third round of PCR
The third round of overlap PCR connects DNA segments 01 with 23, and 45 with 67.

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Figure 3: Gel electrophoresis diagram. Lane 1 is DNA segments 0 to 3. Lane 2 is DNA segments 4 to 7.

Lane 1 shows a correct and bright band for segments 0 to 3. However, although the band for DNA segments 4 to 7 in lane 2 has the correct band, the band is very weak, which means that the amount of this segment is not enough to carry out the construction of plasmid. This is probably because segments 4 to 7 is longer than segments 0 to 3, and the primer is not designed very well. More round of overlap will be needed to obtain more segments of 4 to 7.

The fourth round of PCR
The fourth round of PCR amplifies segments 8 and 9, and overlapped segments 45 with 67 again.

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Figure 4: Gel electrophoresis diagram. Lane 1 to 3 is segments 4567. Lane 4 to 6 is segment 8. Lane 7 to 9 is segments 9.

This result shows that the overlap PCR between segments 45 and 67 was successful. The PCR of segment 9 is also successful. However, there are no clear bands shown in lane 4 to 6, indicating that the PCR of segment 8 is not successful.

Segment 8 was amplified using PCR again.

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Figure 5: Gel electrophoresis diagram. Lane 1 to 3 is segment 8.

This result shows that there are bands on the right position corresponding to the number of base pairs, but they are not clear and bright enough. The product of this round of PCR can be used in the construction of plasmid.

The fifth round of PCR
The fifth round of PCR overlapped segment 67 with 8, and segment 4567 with 8.

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Figure 6: Gel electrophoresis diagram. Lane 1, 3, 5 are segment 45678. Lane 2, 4, 6 are segment 678.

This figure shows that the overlap PCR of segments 67 and 8 was successful, while segments 4567 and 8 was not successful. This is because segment 45678 is too long, which is difficult to carry out accurate overlap PCR.

The sixth round of PCR

The sixth round of overlap PCR connected segments 4567 with 678, which is a modified version of overlap PCR between 4567 and 8 in previous round.

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Figure 7: Gel electrophoresis diagram. Lane 1 is segment 45678.

This result shows that the overlap PCR between segments 4567 and 678 was successful. This is because these two segments have more overlapping base pairs, which ensures higher efficiency during overlap PCR.

Homologous recombination of 0 to 9 fragments

In order to obtain our target plasmid, there are multi-fragment assembly plan A and B. In plan A, PCR amplification products of 0123, 456, 678 and 9 were recovered from gel and the corresponding recombinant plasmids were transformed into competent cells for resistance screening of kana. In plan B, the sequence fragments were 0123, 4567, 8 and 9. Five single colonies in plan A and one single colony in plan B were picked up for further cultivate and plasmid extraction. Identification by electrophoresis showed that plasmid 1,4,5 of plan A and 6 of plan B were the candidate with correct size, in which plasmid 5 of plan A was confirmed by sequencing.

Figure 8: Gel electrophoresis diagram. Line 1-5 is plasmids 1-5 on plan A plate, in which the size of 1, 4, and 5 are correct. Line 6 is plasmid 6 on the plan B plate with correct size. Line 7 is a negative control plasmid.

Figure 9: Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of pYES2-ASADC-SPE1-SpeB. The blast result shows that the plasmid is constructed successfully.

And then, Plasmid 5 was transferred into BY4741 and AQ competent cells. For BY4741, the initial OD600 of seed cells was 0.3, and then cells were collected when OD600 reached 0.9 after 3h of culture. The transformed plasmid pYES2-ASADC-SPE1-SpeB was cultured on YPD20 with hygromycin B plates for 3 days.

Fermentation test

The clones were picked up into YPD20/Hyg test tube for cultivate and activation. In order to observe the growth of fermentation strains, we set up negative control and test group, referred as NC and Test, respectively. Each of them also includes a putrescine production group referred as NC+Arg*10 and Test+Arg*10, respectively, whose medium was supplemented with 10 times Arg.

Figure 10. Growth curve of fermentation strains

As shown in the figure, the growth trend of the four groups was similar. It took 24 hours to reach the stationary phase of growth and this stable period last up to 96 hours. Specifically, the overall growth of NC+Arg*10 and Test+Ar*10g group was a little lower than that of NC and Test group perhaps due to the pressure of high concentration of Arg.

At least 3 repeated small-scale metabolite production tests were carried out in YNB-SC medium (containing 10 times arginine raw material). 2ml supernatant (containing putrescine) of the 48h-metabolites after centrifugation were analyzed by LC-MS.

Figure 11. The peak of putrescine detected by LC-MS.

The results showed that the peak of putrescine appeared at 32 min (as shown in the figure11), indicating that the engineered strain we constructed successfully produced putrescine. It means that our initial plan to produce putrescine based on the engineered strain is possible.

However, the peak area is small, and the output of putrescine is far insufficient from mass production. Thus, we need to make troubleshooting about this problem. It is likely that the protein expression is insufficient and the metabolic pathway is limited. The expression of the three proteins should be optimized in the future, hopefully we will find the expected experiment results to support mass production of putrescine in the near future. If so, we will seek feasible plan to synthesize tropane alkaloids.