Team:SCU-China/Demonstrate
PROJECT
DEMONSTRATION
2019 SCU-China plans to ferment cordycepin and pentostatin together this year. During our fermentation, Cns3 is constitutively expressed and catalyzes the production of pentostatin (PTN), repressing adenosine deaminases (ADA) gradually. We want Cns1 and Cns2 to be expressed when the yeast's growth turn to the plateau stage. And at this stage, the ADA has amostly inhibited by PTN. So, it is the best time for COR production.
As this is the first time we used S. cerevisiae for COR fermentation, one of the most important task to prove that our design can work in yeast.
There are three parts in our design: Cns1 and Cns2 for COR production, Cns3 for PTN production and the delay expression system.
Cns1 and Cns2 for COR production
This is the core of our project. We successfully completed the molecular cloning in the E.coli DH5α and yeast BY4741. And the colony PCR results are shown perfectly. (Figure. 1):
Figure.1 Colony PCR of cns1 and cns2.
(a) Colony PCR of pYES2-Cns1. Lane1 and lane 3-8 show the positive results. (b) Colony PCR of Pyes2-Cns2. Lan5 and lane8 show the positive results.
Then, we did HPLC to verify the synthesis of COR. Luckily, thorugh overcoming many difficulties, we successfully detected the existence of COR (Figure. 2).
Figure. 2 HPLC analysis of COR
Top: Standard sample of 5mM COR.
Middle: Supernatant fraction of Cns1-linker-Cns2 sample.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.
Because of the step-by-step verification of the experiments, we could confirm that the Cns1 and Cns2 can work in the yeast.Fortunately, the fusion protein we designed could worked nicely as well.
Cns3 for PTN production
PTN is a valuable and economic product of our project. Besides, it's also the protector in our project, preventing COR from deamination. We successfully constructed the plasmids of Cns3 and its HisG domain,then we verified their expression and function via RT-qPCR (Figure. 3) and HPLC (Figure. 4). The PTN was clearly detected by HPLC .
Overexpression of Cns3 may consume too much resource and effect the growth of yeast. However，we also need enough PTN to ensure the inhibition of adenosine deaminase. We selected several promoters that have different strength for Cns3. To test the function of these sequences gained from the genome, we chose yeGFP as a reporter (Figure.6). These promoters could work as expected because of the successful expression of yeGFP.
Figure. 3 RT-qPCR of Cns1/Cns1-2/Cns3-HisG-mf/Cns3-HisG-only. No-load is the S. cerevisiae without transformed plasmids. The reference gene is PGK1. The RNA of cns1\cns3 are detected successfully.
Figure. 4 HPLC analysis of PTN
a. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG-MF sample. The strain BY4741 was culturing in YPDG media for 3 days.
b. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of Cns3-hisG ONLY sample. The strain BY4741 was culturing in YPDG media for 3 days.
c. Top: Standard sample of 5mM PTN.
Bottom: Supernatant fraction of BY4741 sample. The strain BY4741 was culturing in YPDG media for 3 days.
Because we wanted to avoid that the constitutive expression of Cns3 consumes so much resource that blocked normal growth of yeast, but we have to guarantee the denature of adenosine deaminase, we selected several different expression level promoters as the upstream sequence of Cns3. (Figure. 5). These sequences could work as promoters as expected because of the successful transcription of Cns3.
Figure. 5 RT-qPCR of constitutive promoters (pPDA1/pTEF1/pTPS1) – Cns3. NC is the S. cerevisiae without transformed plasmids (constitutive promoters – Cns3). The reference gene is PGK1.
Delay expression system
Two key components consist of the delay system: one is the pGAL1, and the other is pMET3. We utilized yeGFP as a reporter to verify their function.
Compared the yeast without galactose, it is obvious that yeast can be induced to express yeGFP (Figure. 6).
Figure. 6 Green fluorescence of S.cerevisiae BY4741 transformed pYES2-yeGFP which was induced by galactose. (a) Fluorescence observation of S.cerevisiae BY4741 transformed pYES2-yeGFP. Series 1: BY4741 WT in YPD medium, Series 2: BY4741 WT in YPG medium, Series 3: BY4741 transformed BY4741 in YPD medium, Series 4: Transformed BY4741 in YPG medium. Visible fluorescence in Series 4. (b) The fluorescence measurement of S.cerevisiaeBY4741 transformed pYES2-yeGFP. YPD: YPD medium, YPG: YPG medium, BY4741 WT: S.cerevisiae, BY4741 wildtype, BY4741 transformed: S.cerevisiae, BY4741 transformed plasmid pYES2-yeGFP, excitation light is 488nm and emission light is 525nm.
As for pMET3, we got this promoter from genome of S. cerevisiae BY4742. To confirm its function, we cultured yeast with and without methionine. It is obvious that without methionine, there is green fluorescence of the yeast while there is no green fluorescence with enough methionine in the culture (Figure. 7).
Figure. 7 The green fluorescence of S. cerevisiae YM4271 transformed pYES2-pMETHIONINE3-yeGFP.
For the further verification of our delay system, we have already constructed the whole gene circuit in a plasmid: pYES2-pMETHIONINE3-GAL4-pGAL1-yeGFP (Figure. 8). We will test the gene circuit in the S. cerevisiae YM4271. For preparation for the test, we tested the growth curve of YM4271 (Figure. 9).
Figure. 8 The double enzyme digestion verification result of pYES2-pMETHIONINE3-GAL4-pGAL1-yeGFP. Marker: Takara DL10000 and Takara DL15000, Lane 1: the plasmid pYES2-pMETHIONINE3-GAL4-pGAL1-yeGFP (10384bp), Lane2: the plasmid pYES2-pMETHIONINE3-GAL4-pGAL1-yeGFP after digesting by BamHI (10384bp).
Figure. 9 Data fitting of experimental data and logistic model and parameter estimates of the model. In the figure, b is the opposite number of growth rate, d is the asymptote and e is the x-axis skewing.
To summary, as the statements above, we think that our design can work basically in yeast, which is of vital importance. And it is worth mention that it is SCU-China first to verify that Cns3 and its HisG domain can work in yeast and attempt to verify Cns1 and Cns2 fusion protein’ function in yeast. Besides the work we did, we do many preparations for future work.