4. Conclusion
In this study, 81 endogenous promoters in Y. lipolytica were
systematically investigated and analyzed. For obtaining the endogenous
promoters, we truncated the 1500 bp upstream sequences before the ATG
site of the desired gene. Specifically, it has been reported that the
core sequences of a promoter in fungi are about 200-300bp. As a result,
our obtained promoters in this study should contain the binding sites of
the transcription factors, thus can accurately reflect some properties
of these endogenous promoters. However, Y. lipolytica could
generate severe fluorescence background, which interferes with the
reliabilities of conventional reporter systems. Specifically, a
luciferase reporter system has been developed in Y. lipolytica ,
which is stable, efficient, and instant.[19]Herein, we chose this luciferase reporter system for the promoter
analysis.
Next, to construct the reporter system plasmids
pYLXP’-Pxx-Nluc , we inserted the promoter
sequences into the chassis plasmid pYLXP’-Nluc , and obtained 82
recombinant promoter plasmids, including the PTEFpromoter. Further, constructed plasmids were transformed into po1g for
the promoter analysis. Specifically, we obtained 15 strong promoters, 41
medium strength promoters and 25 weak promoters. Among them, the most
potent promoter is PMnDH2 with a 1.60-fold strength of
the PTEF promoter, reaching 6.87x107,
which is responsible for transcribing sorbose reductase, catalyzing the
reaction of sorbose to glucitol. Furthermore, the weakest promoter is
PPHO89 (YALI0E23859g ), with a 0.06% strength of
the PTEF promoter, which is responsible for transcribing
sodium-dependent phosphate transporter. These results suggest that we
obtained an endogenous promoter library with a strength spanning from
0.06% to 1.60-fold of the PTEF promoter. Although we
only found several endogenous promoters with higher strength than
PTEF in this study, gene expression levels do not
blindly pursue high strength of the promoter.
We explored the relationship between the promoter activity and time
(Fig. 5), and found that 88.9% promoters had the highest intensity in
the logarithmic growth phase (before 36h). And, 9.9% promoters had the
highest intensity in the stationary phase (48-96 h), while only one
promoter (PPGM2, YALI0E02090g ) reached the
highest transcriptional activity at 72h. In addition, we also found that
the promoter strength is different in the specific metabolic pathways.
For example, promoters in glycolysis are generally more robust. In
conclusion, the promoter library we constructed is significant for
enriching genetic expression elements for Y. lipolytica , and can
be applied to construct microbial cell factories for the
biomanufacturing applications.