Difference between revisions of "Part:BBa K2117000"

Revision as of 16:39, 17 October 2021

Constitutive TEF1 promoter native to Yarrowia lipolytica

Usage and Biology

The translation elongation factor-1α (TEF1) promoter is a strong constitutive promoter. Native for the oleaginous yeast Yarrowia lipolytica.

This promoter is often used for isolation of enzyme genes by expression cloning. However, the promoter is usually not recommended for heterologous production due to early expression of heterologous genes can be detrimental for cell growth.

Characterization

Toulouse_INSA_UPS_2021contributed to the characterization of this part. The constitutive TEF1 promoter had not been characterized before for an expression into S. cerevisiae. The team showed this year that it is functional for expression in S. cerevisiae by expressing the fyn-phCCD1 gene, allowing the production of β-ionone and this molecule was indentified thanks to GC-MS approaches. Check the part of the full construction (BBa_K3930002) for more result details.

Author = ThomasG

Improvement by Evry_Paris-Saclay 2019

This part is functional as a promoter in the oleaginous yeast Yarrowia lipolytica, but it is not compatible with the Type IIS RFC[1000] standard and thus cannot be used in cloning experiments using the Golden Gate technique. As this is a powerful molecular biology technique that allows scarless assembly of a large number of DNA fragments and now it is fully supported by iGEM, we improved this part to make it compatible with the Type IIS RFC[1000] standard.

Thus, we have successfully built 4 versions of the pTef1 promoter: pTef1c (BBa_K2983050), pTef1d (BBa_K2983051), pTef1e (BBa_K2983052) and pTef1f (BBa_K2983053) which are able to drive the expression of a reporter gene in the oleaginous yeast Yarrowia lipolytica at an equivalent strength as this part already present in the iGEM Registry, pTef1a (BBa_K2117000).

Full details are available on the improved pTef1 promoter variants Part's Main Pages: BBa_K2983050, BBa_K2983051, BBa_K2983052 and BBa_K2983053.

Characterization from iGEM20_Calgary

Expression of the reporter gene, hrGFP, under several endogenous Yarrowia lipolytica promoters were tested using flow cytometry. The promoters used in this study include TEF1, EXP, FBA, GPAT, GPD, YAT, and XPR2. Out of the endogenous promoters tested, EXP and TEF1 showed the highest levels of expression for hrGFP, measured as mean fluorescence using a flow cytometer. However, expression levels of these two strong native promoters might sill be too low for some engineering purposes such as metabolic engineering. Therefore, a hybrid TEF1 promoter could be constructed to help improve the expression level of this native promoter.

Hussain et al. conducted a study, examining promoter architecture of various Y. lipolytica promoters. Several promoter components such as upstream activating sequences, proximal promoter sequences, core promoters, and TATA box of endogenous TEF1, POX2, and LEU2 promoters were studied to determine how different promoter elements influence promoter strength and what enables TEF1 promoter to have such high expression levels.

TATA boxes play a crucial role in determining promoter strength as they are responsible for requiring the TFIID general transcription factor. TATA box of TEF1 promoter has a sequence of TATAAAAG and is reported to have one of the highest affinities for TFIID compared to other common TATA box sequences found in Y. lipolytica. To test TATA box strength of various Y. lipolytica promoters, hybrid promoters were constructed using TATA box sequences of TEF1, POX2, and LEU2 promoters. All Hybrid promoters showed strong expression levels, however, the hybrid promoter containing the TEF1 TATA box sequence had the highest levels of expression, indicating that the TEF1 TATA box better facilitates the formation of the transcription machinery compared to other native TATA box sequences.

A similar experiment was performed, testing expression levels of hybrid promoters containing different upstream activation sequences (UAS). These sequences are located upstream of the promoter region and are thought to increase transcriptional activity. The two UAS tested were the UAS1B, a common UAS used to enhance transcription levels of promoters, and the TEF1 UAS. Both UAS showed to increase transcriptional levels but the UASTEF was responsible for a bigger amplification of transcription.

Proximal promoter elements (PPE) are yet another important component of the promoter. These sequences are in the immediate vicinity of the transcription start site and act as recognition sites for various DNA-binding transcription factors. In an experiment to determine the impact of these elements on promoter strength, expression levels of truncated forms of TEF1 promoter that had their PPE sequence removed were compared to normal length TEF1 promoters containing PPE. The results indicate that the TEF1 PPE significantly impacts the promoter strength. Upon removal of PPE, the transcription activity was reduced by more than half. Interestingly, the data suggests that even though the PPE of TEF1 plays a crucial role in promoter strength, its presence is not necessary for promoter function. This is because, in absence of PPE, the promoter still showed a relative amount of transcriptional activity. In contrast, removal of the TATA box in the TEF1 promoter was shown to almost completely diminish transcriptional activity.

Together these results show that components of the TEF1 promoter are among the strongest in the native Y. lipolytica promoters and their inclusion in hybrid promoters can help improve promoter activity. Another experiment was performed to determine what combination of promoter components gives the highest expression level. Not surprisingly, the combination of the core promoter element, PEE, TATA box, and UAS of the TEF promoter produced the highest transcriptional activity which explains why the promoter is considered one of the strongest native promoters of Y. lipolytica.

Based on characterization proved on individual components of TEF1 promoter, alternative forms of this promoter could be synthesized to finetune expression levels. Specific components of the TEF1 promoter such as the UAS and PPE could be included or excluded from the expression construct to help reach the desired transcriptional activity. As shown addition of TEF1 promoter components to other expression constructs could also help improve expression levels.

References

Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Applied and environmental microbiology, 77(22), 7905–7914. https://doi.org/10.1128/AEM.05763-11

Shabbir Hussain, Murtaza, Gambill, Lauren, Smith, Spencer, & Blenner, Mark A. (2016). Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica. ACS Synthetic Biology, 5(3), 213–223. https://doi.org/10.1021/acssynbio.5b00100

Sequence and Features