Team:NJTech China/Engineering



Engineering Success



In short, the purpose of our project this year is to explore the expression ability of natural pheromone-responsive promoters and expand the application range of such promoters through signaling pathway modification. We have designed and characterized artificial promoters according to the role of PREs. Pheromone-inducible promoters show diverse expression capabilities. Promoters with different characteristics can be used for the construction of biosensors and metabolic genetic engineering regulation, and the achievement of intercellular behavior. However, the expensive pheromone limits the application of such promoters in these scenarios. To broaden the application range of pheromone-responsive promoters, we reconstructed the pheromone signaling pathway, trying to build a chassis that responds to cheaper inducers. By reading literature, we learned that Ste5ΔN-CTM protein can activate the MAPK pathway in the absence of pheromone and promote the expression of pheromone-responsive genes. We constructed the Ste5ΔN-CTM strain to enable galactose to induce the expression of mating genes. As a result, the pheromone-responsive promoters can be used for the construction of cell factories.


Research on PREs
Promoter engineering provides sophisticated and diverse expression strategies for metabolic engineering and biosensors. Previous studies have demonstrated the valuable application of promoter engineering in the precise control of gene expression levels1. And the result of promoter modification is relatively stable and not easy to lose during cell culture. During the process of yeast mating, there are a series of promoters induced by pheromone. The strength of pheromone responsive promoters is closely related to the arrangement of pheromone response elements (PREs) in these promoters2.
PREs recruit the transcription factor Ste12 which acts as the major transcriptional activator to activate the promoters in mating genes. Pheromone responsive promoters generally contain multiple copies of PREs. PREs can endow the minimal gal1 core promoter with the ability to respond to pheromone. The sequence, copy number, and orientation of PREs may affect the basal and pheromone responsive expression level of promoters3.
Research has shown that the activity of the promoters strengthens as the number of PREs sites increases, but the association between PRE orientation and promoter strength is not clear. This year our project intends to explore the expression ability of natural pheromone responsive promoters and study the impact of the number and orientation of PREs on it. After reviewing the literature, we selected three promoters, which are pfig1, pprm1, and pfus2, as the research objects. These three promoters contained putative PREs with copy numbers of 5, 3, and 2, respectively4-6. In addition to the difference in the copy number of PREs, they also differ greatly in the orientation and sequence of PREs.

Fig. 1 PREs in pfus2, pprm1 and pfig1. (A) Two PREs in pfus2. (B) Three PREs in pprm1. (C) Five PREs in pfig1.

1.1 Pre-Experiments
First, the growth characteristic of Saccharomyces cerevisiae BY4741 treated by α-factor was evaluated. We conducted halo assay and growth curve measurement. The results showed that α-factor can inhibit the growth of BY4741 on solid and liquid YPD medium. The effect of pheromone in inhibiting growth is positively related to its concentration. These experiments laid an experimental foundation for our later promoter characterization experiments. We chose the concentration with an obvious difference in growth inhibitory effect to set up the subsequent comparative experiments.

Fig. 2 Halo assay of yeast BY4741

Fig. 3 The growth curve of yeast BY4741 treated by different concentrations of pheromone

1.2 Characterization of three natural pheromone responsive promoters
Next, we used GFP reporter genes to characterize pheromone responsive promoters, and constructed engineering yeast strains containing GFP reporter regulated by pprm1, pfig1, or pfus2. After pheromone induction, the intensity of these promoters is reflected by the fluorescence intensity of GFP which is quantified by flow cytometry. Then, we analyzed the intensity of promoters at different pheromone concentrations, demonstrating the diverse expression capability of pheromone responsive promoters. When OD=0.6, α-factor was added for induction. Results showed that the pheromone has a significant inducing effect on these three promoters. Within a certain range, which is among 1-5 , the higher the concentration of pheromone, the more obvious the induction effect is. Comparing the GFP expression level of these three promoters, pfus2 is the strongest, followed by pfig1, and pprm1 is the weakest.

Fig. 4 The fluorescence intensity of GFP expressed by different promoters induced by different concentrations of pheromone

1.3 New findings from promoter characterization experiments
We summarized several conclusions that have not been reported before. According to previous predictions, the expression level of pfus2 should be the weakest, but the experimental results showed that the intensity of pfus2 was higher than pprm1. According to the literature and database, the two PRE sites of pfus2 have opposite directions, while the three PRE sites of pprm1 are in the same direction. It has been reported that the expression level of the promoter with the PRE sites in the "tail-to-tail" orientation is higher than that of the promoter with the PRE sites in the "same direction" orientation, and our experimental results are consistent with this. The specific influence caused by different PRE orientations needs to be investigated and verified by further experiments.

1.4 Modification of natural promoters
Based on the characterization results, we found that although pprm1 possesses three PREs, it has a low induced expression level which indicates a good potential for modification. The background expression level of this promoter is low which means it can be used to express some proteins that require lower concentrations in the early stage. Based on the literature, we learned the general effect of PREs on promoter strength. For example, an increase in the copy number of PREs can enhance the induced expression level of the promoter. And a PRE which is aligned in the direction of the promoter may be more effective than a PRE which is in the opposite direction of the promoter3. In the first modification scheme, we doubled the 3 × PRE sequence in natural pprm1. The modified pprm1 is referred to as pprm1 Ultra which contains 6 × PREs. In the second modification scheme, we changed the orientation of the 3 × PREs in natural pprm1. The modified promoter is referred to as pprm1 Pro which contains three PREs oriented in the same direction as the promoter. As a consequence, the two modified promoters exhibit characteristics remarkably different from the wild type. Unexpectedly, the activity of pprm1 is higher than that of the pprm1 Pro and that of the pprm1 Ultra under the treatment of high concentration pheromones. The activity of the pprm1 Ultra is higher than that of the pprm1 Pro, which means that the orientation of the PRE site on the promotor is critical to the expression level of the promoter. Although the experimental results did not meet our expectations, this result still provided some interesting conclusions. e.g.（Please see the Gold Award section for details：http://parts.igem.org/wiki/index.php?title=Part:BBa_K3384314）

Fig. 5 Improvement of pprm1

Fig. 6 The fluorescence intensity of GFP expressed by different modified pprm1 induced by different concentrations of pheromone

Taking the experimental period and other factors into account, we only selected a few pheromone concentrations gradient for the experiment. At the same time, our experimental sample size is limited.

1.5 The application of Mathematical models in our project
Mathematical models are another important tool for characterizing promoters. In our project, neural network algorithms can predict the expression level of pheromone-inducible promoters. The molecular dynamics model guides the design of experimental conditions and is the basis for obtaining a relatively accurate pheromone concentration profile of the promoter through as few experiments as possible.（ https://2020.igem.org/Team:NJTech_China/Model ）


Construction of the Ste5ΔN-CTM strain

2.1 Inspiration and design


In the course of the experiment, we found that the price of pheromone is much higher than that of the galactose. The price of 10mg of the pheromone can buy nearly 800g of the galactose. We hope to modify the yeast pheromone signaling pathway so that other inducers can activate the expression of pheromone-responsive promoters, thereby cutting the expenses in some biological experiments. The pheromone signaling pathway is a process of coupling between G protein and MAPK pathway. Among them, Ste5 plays an important role responsible for associating the G protein with the MAPK component in the signaling pathway. It can be seen that the modular structure of Ste5 provides possibility for the modification of the signaling pathway. Inspired by this, we intended to construct an engineering S. cerevisiae strain as a chassis cell for research, in which the pheromone responsive promoters can be induced by the cheap inducer galactose. After reviewing the literature, we found a Ste5 mutant called the Ste5ΔN-CTM7. The 214 amino acids at the N-terminus of Ste5 contain membrane binding sites and mediate the membrane localization of Ste5 under the control of Gβγ. By deleting the N-terminal sequence, the activation of Ste5 is independent of G protein. The C-terminal transmembrane domain of Snc2 was added at the Ste5ΔN C-terminus in order to constitutively recruit Ste5 to the plasma membrane. Ste5ΔN-CTM protein can activate the MAPK pathway in the absence of pheromone and promote the expression of pheromone-responsive genes.

2.2 Construction of Δste5 strains
We aim to eliminate the interference of natural Ste5 on the modified signaling pathway. Initially, we learned that the CRISPR-Cas9 system is widely applied to gene knockout in S. cerevisiae, so we decided to try the CRISPR-Cas9-based gene knockout technology. Firstly, we used ChopChop and Benchling to design gRNA, constructed recombinant plasmids containing gRNA and carried out yeast transformation. But the PCR results showed that the gene knockout was unsuccessful. We sequenced the pCas9 plasmid and found some problems with its sequence. We will analyze the specific reason for failure and modify the plasmid.
To achieve the Δste5 strains, we used homologous recombination for gene knockout. Through nucleic acid electrophoresis and sequencing, we filtered out the Δste5 strain. We named it as ste5Δ-loxp-1.

Fig. 7 Nucleic acid electrophoresis verification of strain ste5Δ-loxp-1

To further verify the knockout of ste5, we conducted a Halo assay experiment. According to the related literature, if the haploid yeast lacks the Ste5 protein, the yeast will not be able to respond to the pheromone, and the cell growth will not be inhibited, failing to form a halo on the plate. The ste5Δ-loxp-1 strain did not form a halo on the plate. This result is in line with the description of the ste5-deficient yeast strain in the literature.

Fig. 8 Halo assay of strain ste5Δ-loxp-1

2.3 The construction of pheromone-independent Ste5 highly active mutant
After the construction of the Δste5 strain, we introduced the pgal1-Ste5ΔN-CTM gene into it, and named it the pheromone-independent Ste5 highly active mutant, PIDS for short. The engineered yeast can transform from the vegetative phase to the mating phase under the induction of galactose. To further characterize the induction of PIDS by galactose, we plan to transform the pheromone responsive GFP reporter gene into PIDS to quantitatively evaluate the signal transduction level of this mutant. We spread the PIDS strain on a plate with galactose as the sole carbon source.
According to relevant literature, the strain should not be able to grow on the plate. Our experimental results are consistent with the theory. The results of the experiment are shown in Figure 9. The left side of the figure is the PIDS strain and the right side is the BY4741 strain. It can be clearly seen that there is no colony growth on the left side and the yeasts on the right side grow well.

Fig. 9 The growth of the Ste5 ΔN-CTM strain and BY4741wild type strain on the plate with galactosyl as the sole carbon source

Fig. 10 The growth of the Ste5ΔN-CTM strain and BY4741 wild-type strain on the plate with glucose as the sole carbon source

As shown in figure 9, the left side of the plate is the PIDS strain and the right side is the BY4741 strain. It can be clearly seen that there is no colony growth on the left side and the yeast on the right side grows well. Meanwhile, as shown in figure 10, on SC complete medium, both PIDS strain and BY4741 strain grew well.


References

[1] Xu, N., Wei, L., and Liu, J. (2019) Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis, World J Microbiol Biotechnol 35, 33.
[2] Sengupta, P., and Cochran, B. H. (1990) The PRE and PQ box are functionally distinct yeast pheromone response elements, Mol Cell Biol 10, 6809-6812.
[3] Su, T. C., Tamarkina, E., and Sadowski, I. (2010) Organizational constraints on Ste12 cis-elements for a pheromone response in Saccharomyces cerevisiae, FEBS J 277, 3235-3248.
[4] Elion, E. A., Trueheart, J., and Fink, G. R. (1995) Fus2 Localizes near the Site of Cell-Fusion and Is Required for Both Cell-Fusion and Nuclear Alignment during Zygote Formation, Journal of Cell Biology 130, 1283-1296.
[5] Erdman, S., Lin, L., Malczynski, M., and Snyder, M. (1998) Pheromone-regulated genes required for yeast mating differentiation, J Cell Biol 140, 461-483.
[6] Heiman, M. G., and Walter, P. (2000) Prm1p, a pheromone-regulated multispanning membrane protein, facilitates plasma membrane fusion during yeast mating, J Cell Biol 151, 719-730.
[7] Lamson, R. E., Takahashi, S., Winters, M. J., and Pryciak, P. M. (2006) Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity, Curr Biol 16, 618-623.