Characterization and design of promoters with different strength and sensitivity to
appropriate inducing conditions in Yarrowia lipolytica are crucial for its synthetic
biological applications. Previous works either focus on improving one single constitutive
promoter (Paris-Saclay 2021) or characterizing endogenous promoter regions (NNU 2022), and
none of them reported the inducibility of Yarrowia lipolytica promoters. Based on the
sequence and characterizations done, we proposed several design strategies to optimize
Yarrowia lipolytica promoters pCIT1 (BBa_K3868074). The promoter was chosen because
it is a
medium-strong promoter induced by nitrogen starvation[1], which well suits our stage control
strategy (Engineering Cycle 3.1) .
It also serves as a good demonstration of improving
other Yarrowia lipolytica endogenous promoters for our use, especially inducible ones as
there are few existing researches focused on those promoters.
At first, our idea is to make use of the promoter library in a relatively well-studied
chassis, e.g., S. cerevisiae, and make modifications and hybridization to enable its
corresponding function in Yarrowia lipolytica. However, in our interview with
Prof. Yong
Lai, he and his PhD candidate student pointed out the poor transferability of
promoters
across species, and the huge basis of data needed for properly predicting promoter
functions. The transcription factors and conserved promoter regions in the two species can
be very different, as Y. lipolytica and S. cerevisiae were evolutionarily
branched over 300
million years ago. Therefore, we decided to focus on the endogenous promoters.
Addition of an intron sequence
Graph 1. Coding gene under a core promoter with intron
Including a native intron sequence after the traditionally identified promoter region
that
ends at the start codon sometimes can significantly improve the promoter strength[2]. In our
project, we used the TEF promoter with the intron sequence which can increase its
strength
by 17 folds, tested by eGFP fluorescence[3]. The
intron
sequence of one promoter is also
possible to function under another.
Amplification of existing promoters
Graph 2. Coding gene under a core promoter with tandem repeats of
UAS
One of the most common Yarrowia lipolytica strong promoters is the hybrid promoter
Hp4d,
which contains 4 tandem repeats of the upstream activating sequence 1B (UAS1B), followed
by
a LEU2 core promoter[4]. UAS1B was identified from
upstream of the EXP2 promoter, and it may
change inducibility of the core promoter [5].
Finally, combining the above 2 strategies, we can obtain a general design for tuning
promoter
strength in Yarrowia lipolytica. The compatibility of the 2 strategies will be
tested
in the
future.
Graph 3. Coding gene under a core promoter with both intron and
UAS
Identification of the core promoter region
The main limitation of existing endogenous promoters is that the exact promoter region is
not
identified, instead, people simply choose 1500 bp or 1000 bp upstream of the coding gene
as
the functional promoter region [1], while further
characterization is only done for limited
promoters, among which constitutive ones account for a large proportion. Therefore with
existing clues from the binding sites and a dichotomic strategy, we will truncate the
promoters from the 5’ end to test its strength and sensitivity by comparing to original
ones
[8].
[1] Chang Wang, Mingxin Lin, Zhiliang Yang, Xueyao Lu, Yinfang Liu, Huizhi Lu, Jiang Zhu,
Xiaoman Sun, Yang Gu (2023). Characterization of the endogenous promoters in Yarrowia
lipolytica for the biomanufacturing applications. Process Biochemistry, Volume 124, 245-252.
https://doi.org/10.1016/j.procbio.2022.11.023
[2] Dietrich et al (2023). Refactoring the architecture of a polyketide gene cluster
enhances docosahexaenoic acid production in Yarrowia lipolytica through improved expression
and genetic stability. Microbial Cell Factories, 22:199.
https://doi.org/10.1186/s12934-023-02209-9
[3] Mitchell Tai, Gregory Stephanopoulos (2013). Engineering the push and pull of lipid
biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metabolic
Engineering, Volume 15, 1-9.
https://doi.org/10.1016/j.ymben.2012.08.007
[4] Blazeck J. Liu L. Redden H, Alper H (2011). Tuning Gene Expression in Yarrowia
lipolytica by a Hybrid Promoter Approach. Appl Environ Microbiol, Vol.77, 22.
https://doi.org/10.1128/AEM.05763-11
[5] Blazeck, J., Reed, B., Garg, R. et al (2013). Generalizing a hybrid synthetic promoter
approach in Yarrowia lipolytica . Appl Microbiol Biotechnol 97, 3037–3052.
https://doi.org/10.1007/s00253-012-4421-5
[6] Zhao, Y., Liu, S., Lu, Z. et al (2021). Hybrid promoter engineering strategies in
Yarrowia lipolytica: isoamyl alcohol production as a test study. Biotechnol Biofuels 14,
149.
https://doi.org/10.1186/s13068-021-02002-z
[7] Murtaza Shabbir Hussain, Lauren Gambill, Spencer Smith, and Mark A. Blenner (2016).
Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica. ACS Synthetic
Biology, 2016, 5 (3), 213-223.
https://doi.org/10.1021/acssynbio.5b00100
[8] Benjamin Ouellet, A.M. Abdel-Mawgoud (2023). Strong expression of Cas9 under a new
3′-truncated TEF1α promoter enhances genome editing in Yarrowia lipolytica. Current Research
in Biotechnology, Volume 6, 100147.
https://doi.org/10.1016/j.crbiot.2023.100147
